CN106998665A - 单倍体植物的产生 - Google Patents
单倍体植物的产生 Download PDFInfo
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Abstract
本发明涉及非转基因和转基因植物,优选农作物,其包含导致着丝粒组蛋白H3(CENH3)的CATD结构域、优选CATD结构域的环1或者α2‑螺旋中氨基酸序列改变的突变,所述植物具有单倍体诱导物的生物活性。进一步地,本发明提供产生本发明的植物的方法,可通过将本发明的植物与野生型植物杂交获得的单倍体和双单倍体植物,以及促进细胞质交换的方法。
Description
本发明涉及非转基因和转基因植物、优选农作物,其包含导致着丝粒组蛋白H3(CENH3)的CATD结构域内、优选在CATD结构域的环1和/或α2-螺旋内的氨基酸改变的至少一个突变,所述植物具有单倍体诱导物生物活性。进一步地,本发明提供产生本发明植物的方法和可通过将本发明植物与野生型植物杂交获得的单倍体和双单倍体植物,以及促进细胞质交换的方法。
单倍体的产生和使用是改良栽培植物的最有效的生物技术手段之一。单倍体对于培育者的优势是在双单倍体化,产生双单倍体植物之后的第一代中已经可以实现纯合性,不需要获得高度纯合性所要求的若干回交世代。进一步地,单倍体在植物研究和培育中的价值在于双单倍体的生成细胞是减数***的产物,由此所得群体组成了多样性重组体及同时遗传固定的个体的集合。因此,双单倍体的产生不仅提供了极有用的遗传变异性(进行选择用于作物改良),而且还提供了有价值的方式以产生作图群体、重组近交系以及直接纯合(instantly homozygous)的突变体以及转基因株系。
单倍体可以通过体外或体内方法获得。然而,许多物种和基因型对于这些方法是顽固的(recalcitrant)。或者,通过交换着丝粒特异性组蛋白H3变体(CENH3,也称作CENP-A)的N末端区并将其与GFP融合(“GFP-尾部交换”(GFP-tailswap)CENH3),显著改变所述蛋白质,在模式植物拟南芥(Arabidopsis thaliana)中产生单倍体诱导物株系(Ravi andChan,Nature,464(2010),615-618;Comai,L,"Genome elimination:translating basicresearch into a future tool for plant breeding.",PLoS biology,12.6(2014))。CENH3蛋白是H3组蛋白的变体,其是活性着丝粒的着丝点复合物的成员。使用这些“GFP-尾部交换”单倍体诱导物株系,当单倍体诱导物植物与野生型植物杂交时在后代中发生单倍体化。令人感兴趣地,单倍体诱导物株系在自交时是稳定的,提示在产生杂交胚中修饰的着丝粒与野生型着丝粒之间的竞争导致诱导物亲本的着丝粒失活,并因此导致单亲本染色体消除。结果,含有改变的CENH3蛋白的染色体在早期胚胎发育期间丧失,产生仅含有野生型亲本染色体的单倍体后代。
因此,单倍体植物可以通过将作为单倍体诱导物的“GFP-尾部交换”转基因植物与野生型植物杂交而获得。然而,如上所述,这种技术需要大量改变CENH3蛋白质,且所述植物包含异源转基因,这样由于增加了关于基因工程化作物的公众阻碍而存在经济学问题。
因此,本发明的一个目的是克服前述问题及特别提供了替代的单倍体诱导物植物,其不包含对其CENH3蛋白质的大量修饰和/或不是基因工程化的。
这个问题通过独立权利要求的主题得以解决,特别是由具有单倍体诱导物生物活性及包含编码具有CATD结构域的着丝粒组蛋白H3(CENH3)的核苷酸序列的植物而解决,其中所述核苷酸序列包含导致在CATD结构域中CENH3蛋白质的氨基酸序列改变的突变,所述改变赋予单倍体诱导物生物活性。CENH3蛋白质的CATD结构域对应于来源于拟南芥的SEQID No.38的位置113-155的氨基酸序列,和/或CENH3蛋白质的CATD结构域由对应于来源于拟南芥的SEQ ID No.37的位置337-465的核苷酸序列编码。拟南芥序列仅作为参考,并不将本发明限于具体的拟南芥序列。由于高水平的保守,本领域技术人员在任何其它植物材料或植物物种中能发现对应于拟南芥序列的核苷酸序列和氨基酸序列。在本发明中,术语“改变”是指对CENH3蛋白质的氨基酸序列的任何修饰(包括多重修饰),这是由在编码着丝粒组蛋白H3(CENH3)的核苷酸序列中的至少一个突变所致。核苷酸序列可以是基因组DNA或者CENH3基因的cDNA。改变可以是取代一或多个氨基酸、***一或多个氨基酸或者缺失一或多个氨基酸。能改变蛋白质CENH3的氨基酸序列的在DNA水平的突变可以是导致氨基酸取代或者终止密码子的点突变、移动了CENH3基因的读框的***或缺失,或者是在剪接位点的突变。
在一个优选的实施方案中,导致氨基酸取代的突变位于CATD结构域的环1内。环1对应于来源于拟南芥的SEQ ID No.38所示位置114-126的氨基酸序列,和/或环1由对应于来源于拟南芥的SEQ ID No.37的核苷酸位置340-378的核苷酸序列编码。所述拟南芥序列仅作为参考,不将本发明限于特异性的拟南芥序列。由于高水平的保守,本领域技术人员在任何其它植物材料或植物物种中能发现对应于拟南芥序列的核苷酸序列和氨基酸序列。
在另一个优选的实施方案中,导致氨基酸取代的至少一个突变位于CATD结构域的α2螺旋内。α2-螺旋对应于来源于拟南芥的SEQ ID No.38所示位置127-155的氨基酸序列,和/或α2-螺旋由对应于来源于拟南芥的SEQ ID No.37所示核苷酸位置379-465的核苷酸序列编码。所述拟南芥序列仅作为参考,不将本发明限于特异性的拟南芥序列。由于高水平的保守,本领域技术人员在任何其它植物材料或植物物种中能发现对应于拟南芥序列的核苷酸序列和氨基酸序列。
CENH3蛋白质是H3组蛋白的变体,其是活性着丝粒的着丝点复合物的成员,即在细胞***期间纺锤丝附着的染色体上的蛋白质结构。基本上,CENH3蛋白质特征在于不形成严格二级结构的可变的尾部结构域,及由三个α-螺旋区(称作α1-α3,通过两个环部分连接)组成的保守的组蛋白折叠结构域。高度保守的CATD结构域(CENP-A靶向结构域)位于组蛋白折叠结构域内,其是由部分α1-螺旋、完整的α2-螺旋及连接环1组成。保守的CATD结构域为伴侣蛋白荷载CENH3所要求,因此对于其着丝点定位和着丝粒功能至关重要。
本发明人令人惊奇地发现可以通过取代保守CATD结构域内、特别是CENH3蛋白质的环1或α2-螺旋中的一个单一氨基酸而获得具有产生单倍体后代能力即单倍体诱导物功能的植物。有利地,这可以通过转基因以及非转基因方法而实现。由于违反基因改造的生物体(GMO)的规定以及增加公众对于基因改造的生物体(GMO)或者通过GMO产生的植物特别是用于人类消费的作物的抗拒以及大量的市场授权过程(包括这种GMO的严格安全性评价)的巨额成本,优选非转基因方法。
本发明提供包含且表达具有CATD结构域的CENH3蛋白质的植物,其中在CATD结构域中,特别是在环1或者α2-螺旋中,最优选在分别具有SEQ ID No.49或1的共有序列的环1或者α2-螺旋中,在所述植物的内源性编码的CENH3蛋白质中发生的氨基酸缺失或者由另一氨基酸取代。这种改变可赋予植物单倍体诱导物生物活性。
在优选的实施方案中,部分涉及包含编码具有CATD结构域的着丝粒组蛋白H3(CENH3)的核苷酸序列的植物,其中部分编码CATD结构域的核苷酸序列包含突变,及其中所述突变导致在环1中CENH3蛋白质的CATD结构域中氨基酸序列改变,其a)由对应于来源于拟南芥的SEQ ID No.37所示核苷酸位置340-378的核苷酸序列编码,其对应于来源于拟南芥的SEQ ID No.38的位置114-126的氨基酸序列,或者由对应于来源于甜菜(Beta vulgaris)的SEQ ID No.60的核苷酸位置271-306的核苷酸序列编码,其对应于来源于甜菜的SEQ IDNo.61的位置91-102的氨基酸序列,或者由对应于来源于甘蓝型油菜(Brassica napus)的SEQ ID No.51的核苷酸位置346-384的核苷酸序列编码,其对应于来源于甘蓝型油菜的SEQID No.52的位置116-128的氨基酸序列,或者由对应于来源于玉米(Zea mays)的SEQ IDNo.57的核苷酸位置280-318的核苷酸序列编码,其对应于来源于玉米的SEQ ID No.58的位置94-106的氨基酸序列,或者由对应于来源于高粱(Sorghum bicolor)的SEQ ID No.54的核苷酸位置280-318的核苷酸序列编码,其对应于来源于高粱的SEQ ID No.55的位置94-106的氨基酸序列,或者由对应于来源于大麦(Hordeum vulgare)的SEQ ID No.33(βCENH3)的核苷酸位置208-264的核苷酸序列编码,其对应于来源于大麦的SEQ ID No.34(βCENH3)的位置70-88的氨基酸序列,或者具有SEQ ID No.49的共有序列,及b)位于如上述CENH3蛋白质的CATD结构域内,或者所述突变导致α2-螺旋中CENH3蛋白质的CATD结构域中氨基酸序列改变,其a)由对应于来源于拟南芥的SEQ ID No.37的核苷酸位置379-465的核苷酸序列编码,其对应于来源于拟南芥的SEQ ID No.38的位置127-155的氨基酸序列,或者由对应于来源于甜菜的SEQ ID No.60的核苷酸位置307-393的核苷酸序列编码,其对应于来源于甜菜的SEQ ID No.61的位置103-131的氨基酸序列,或者由对应于来源于甘蓝型油菜的SEQID No.51的核苷酸位置385-471的核苷酸序列编码,其对应于来源于甘蓝型油菜的SEQ IDNo.52的位置129-157的氨基酸序列,或者由对应于来源于玉米的SEQ ID No.57的核苷酸位置319-405的核苷酸序列编码,其对应于来源于玉米的SEQ ID No.58的位置107-135的氨基酸序列,或者由对应于来源于高粱的SEQ ID No.54的核苷酸位置319-405的核苷酸序列编码,其对应于来源于高粱的SEQ ID No.55的位置107-135的氨基酸序列,或者由对应于来源于大麦的SEQ ID No.33(βCENH3)的核苷酸位置265-351的核苷酸序列编码,其对应于来源于大麦的SEQ ID No.34(βCENH3)的位置89-117的氨基酸序列,或者具有SEQ ID No.1的共有序列,及b)位于如上述CENH3蛋白质的CATD结构域内。因此,优选所述改变位于结构域的环1或α2-螺旋中。CATD结构域的非突变的环1在植物物种中是高度保守的,长度为13个氨基酸,从位置1开始至位置13结束。在本发明中,关于环1或者下述SEQ ID No.49的共有序列给出的任何氨基酸位置均是关于这个编号***。优选地,非突变的环1呈现出表1中示出的氨基酸序列。
表1:在CENH3蛋白质的环1中的确定的(specified)氨基酸
在环1内的位置 | 氨基酸 |
1 | T、S或A |
2 | H、Q、N、A、Y、F、G、D或E |
3 | M、Q、I、F、Y、A、E、N、R、L、H或G |
4 | L、F、V、I或Y |
5 | A、T、S、C或M |
6 | P、N、D、R、A、T、F、R、H、S或K |
7 | X |
8 | Q、Y、D、K、R、E、G、S、P、H、N或A |
9 | I、V或P |
10 | N、G、T、E,或S |
11 | R或P |
12 | W或Y |
13 | T、Q或S |
更优选地,环1具有SEQ ID No.49所示共有序列,其是:
TNFLA PXEVT RWT
5 10 13
如上所示,环1包含不确定的氨基酸(以X表示)和确定的氨基酸[以单字母代码表示]。
CATD结构域的非突变的α2-螺旋在植物物种中是高度保守的其长度为29个氨基酸,从位置1开始至位置29结束。在本发明中,关于α2-螺旋或下述SEQ ID No.1所示共有序列的氨基酸位置是关于这个编号***。优选地,非突变的α2-螺旋呈现出如下表2所示氨基酸序列。
表2:CATD结构域的α2-螺旋中的确定的氨基酸
更优选地,α2-螺旋具有SEQ ID No.1所示共有序列,其是:
如上所示,α2-螺旋包含确定的氨基酸(以单字母代码表示)。
根据本发明的一个优选实施方案,导致CENH3蛋白质的CATD结构域中氨基酸序列改变的突变可以产生具有产生单倍体后代能力的想要的植物,所述突变是如在表2或SEQID No.1或者在表1或SEQ ID No.49中限定的任何不确定的氨基酸或者确定的氨基酸的突变,优选氨基酸的取代或缺失。
如在表1或SEQ ID No.49中给出的不确定的氨基酸是这样的氨基酸,尽管其在一组特定植物物种、在特定植物属或者在一特定植物物种中是确定的,但是在更大范围的植物物种中不是保守的。因此,在一组特定的植物物种中、在特定的植物属中或者在一特定植物物种中,SEQ ID No.49所示或表1给出的不确定的氨基酸是明确限定的特定的氨基酸,然而在其它植物物种中相同位置可能未发现。因此,SEQ ID No.49或者如表1所示的不确定的氨基酸的氨基酸取代是指在植物中、即在特定植物物种中,特定的但非保守的氨基酸由另一氨基酸取代,所述另一氨基酸与在这组特定植物物种、在这个特定的植物属或者在这个特定的植物物种中在所述植物物种的内源性编码的天然CENH3蛋白质中在该位置天然存在氨基酸不同。此外,不确定的氨基酸以及确定的氨基酸可以是蛋白质折叠或蛋白质稳定性必需的氨基酸。这种氨基酸的改变可导致具有削弱的稳定性或不正确折叠的突变体CENH3产生。
在表1和表2中示出的确定的氨基酸及SEQ ID No.49和1所示特定氨基酸是在广泛的植物物种中存在的那些氨基酸,优选如下文列出的那些,因此,其是非常保守的。
在优选的实施方案中,SEQ ID No.49或1所示共有序列是来源于选自如下一组的物种的环1和α2-螺旋的序列编辑的:大麦、Hordeum bulbusom、高粱、甘蔗(Saccharumofficinarium)、玉米、小米(Setaria italica)、小粒野生稻(Oryza minuta)、水稻(Orizasativa)、澳洲野生稻(Oryza australiensis)、野生稻(Oryza alta)、小麦(Triticumaestivum)、黑麦(Secale cereale)、苹果(Malus domestica)、二穗短柄草(Brachypodiumdistachyon)、Hordeum marinum、山羊草(Aegilops tauschii)、Daucus glochidiatus、甜菜、Daucus pusillus、Daucus muricatus、胡萝卜(Daucus carota)、巨桉(Eucalyptusgrandis)、美花烟草(Nicotiana sylvestris)、绒毛烟草(Nicotiana tomentosiformis)、烟草(Nicotiana tabacum)、番茄(Solanum lycopersicum)、马铃薯(Solanum tuberosum)、中果咖啡(Coffea canephora)、葡萄(Vitis vinifera)、Erythrante guttata、Genliseaaurea、黄瓜(Cucumis sativus)、桑树(Morus notabilis)、Arabidopsis arenosa、深山南芥(Arabidopsis lyrata)、拟南芥、须弥芥(Crucihimalaya himalaica)、卵叶须弥芥(Crucihimalaya wallichii)、弯曲碎米荠(Cardamine flexuosa)、北美独行菜(Lepidiumvirginicum)、荠菜(Capsella bursa pastoris)、Olmarabidopsis pumila、硬毛南芥(Arabis hirsute)、甘蓝型油菜、甘蓝(Brassica oeleracia)、芜菁(Brassica rapa)、萝卜(Raphanus sativus)、芥菜(Brassica juncea)、黑芥菜(Brassica nigra)、芝麻菜苜蓿亚种(Eruca vesicaria subsp.Sativa)、甜橙(Citrus sinensis)、麻风树(Jatrophacurcas)、毛果杨(Populus trichocarpa)、蒺藜苜蓿(Medicago truncatula)、Ciceryamashitae、Cicer bijugum、鹰嘴豆(Cicer arietinum)、野生鹰嘴豆(Cicerreticulatum)、Cicer judaicum、Cajanus cajanifolius、蔓草虫豆(Cajanusscarabaeoides)、菜豆(Phaseolus vulgaris)、大豆(Glycine max)、紫云英(Astragalussinicus)、百脉根(Lotus japonicas)、夏堇(Torenia fournieri)、洋葱(Allium cepa)、葱(Allium fistulosum)、蒜(Allium sativum)和韭菜(Allium tuberosum)。
在特别优选的实施方案中,突变导致如表1或表2所示的确定的氨基酸的取代或缺失。因此,本发明的植物包含如表1或2所示的确定的氨基酸,即在表1或表2中保守及命名的那些氨基酸的至少一个取代或缺失。
如表1中所示的确定的氨基酸的取代或缺失应是指选自如下组的氨基酸的取代或缺失:
a)在位置1的苏氨酸、丝氨酸或者丙氨酸,
b)在位置2的组氨酸、谷氨酰胺、天冬酰胺、丙氨酸、酪氨酸、苯丙氨酸、甘氨酸、天冬氨酸或谷氨酸,
c)在位置3的甲硫氨酸、谷氨酰胺、异亮氨酸、苯丙氨酸、酪氨酸、丙氨酸、谷氨酸、天冬酰胺、精氨酸、亮氨酸、组氨酸或甘氨酸,
d)在位置4的亮氨酸、苯丙氨酸、缬氨酸、异亮氨酸或酪氨酸,
e)在位置5的丙氨酸、苏氨酸、丝氨酸、半胱氨酸或甲硫氨酸,
f)在位置6的脯氨酸、天冬酰胺、天冬氨酸、精氨酸、丙氨酸、苏氨酸、苯丙氨酸、精氨酸、组氨酸、丝氨酸或赖氨酸,
g)在位置8的谷氨酰胺、酪氨酸、天冬氨酸、赖氨酸、精氨酸、谷氨酸、甘氨酸、丝氨酸、脯氨酸、组氨酸、天冬酰胺或丙氨酸,
h)在位置9的异亮氨酸、缬氨酸或脯氨酸,
i)在位置10的天冬酰胺、甘氨酸、苏氨酸、谷氨酸或丝氨酸,
j)在位置11的精氨酸或脯氨酸,
k)在位置12的色氨酸或酪氨酸,及
l)在位置13的苏氨酸、谷氨酰胺或丝氨酸。
如表2中所示确定氨基酸的取代或缺失应是指选自如下组的氨基酸的取代或缺失:
a)在位置1的丙氨酸、脯氨酸、缬氨酸或亮氨酸,
b)在位置2的谷氨酸、天冬氨酸、谷氨酰胺、组氨酸或亮氨酸,
c)在位置3的丙氨酸,
d)在位置4的亮氨酸或缬氨酸,
e)在位置5的缬氨酸、亮氨酸、甲硫氨酸、异亮氨酸、精氨酸、酪氨酸或苏氨酸,
f)在位置6的丝氨酸或丙氨酸,
g)在位置7的异亮氨酸或亮氨酸,
h)在位置8的谷氨酰胺,
i)在位置9的谷氨酸,
j)在位置10的丙氨酸或丝氨酸,
k)在位置11的丙氨酸或苏氨酸,
l)在位置12的谷氨酸,
m)在位置13的天冬氨酸、天冬酰胺、苯丙氨酸、异亮氨酸或酪氨酸,
n)在位置14的酪氨酸、苯丙氨酸或组氨酸,
o)在位置15的亮氨酸、异亮氨酸或缬氨酸,
p)在位置16的缬氨酸或异亮氨酸,
q)在位置17的甘氨酸、精氨酸、谷氨酸、组氨酸、天冬酰胺、苏氨酸、谷氨酸、天冬氨酸或谷氨酰胺,
r)在位置18的亮氨酸、甲硫氨酸或异亮氨酸,
s)在位置19的苯丙氨酸、甲硫氨酸或亮氨酸,
t)在位置20的丝氨酸、谷氨酸、天冬氨酸或甘氨酸,
u)在位置21的天冬氨酸、甲硫氨酸、缬氨酸、天冬酰胺、谷氨酸、丙氨酸、精氨酸、赖氨酸,
v)在位置22的丝氨酸、甘氨酸、丙氨酸或苏氨酸,
w)在位置23的甲硫氨酸、色氨酸、天冬酰胺或组氨酸,
x)在位置24的亮氨酸或组氨酸,
y)在位置25的半胱氨酸或亮氨酸,
z)在位置26的丙氨酸或苏氨酸,
aa)在位置27的亮氨酸或异亮氨酸,
bb)在位置28的组氨酸,及
cc)在位置29的丙氨酸或丝氨酸。
在一特别优选的实施方案中,至少一个突变导致SEQ ID No.49的确定的氨基酸的取代或缺失。因此,本发明的植物包含SEQ ID No.49的确定的氨基酸,即在SEQ ID No.49的共有序列中高度保守及命名的那些氨基酸的至少一个取代或缺失。SEQ ID No.49的确定的氨基酸的取代或缺失应是指选自如下组的氨基酸的取代或缺失:
a)在位置1的苏氨酸,
b)在位置2的天冬酰胺,
c)在位置3的苯丙氨酸,
d)在位置4的亮氨酸,
e)在位置5的丙氨酸,
f)在位置6的脯氨酸,
g)在位置8的谷氨酸,
h)在位置9的缬氨酸,
i)在位置10的苏氨酸,
j)在位置11的精氨酸,
k)在位置12的色氨酸,及
l)在位置13的苏氨酸。
在一特别优选的实施方案中,突变导致SEQ ID No.1的确定的氨基酸的取代或缺失。因此,本发明的植物包含SEQ ID No.1的确定的氨基酸,即在SEQ ID No.1的共有序列中高度保守及命名的那些氨基酸的至少一个取代或缺失。SEQ ID No.1的确定的氨基酸的取代或缺失应是指选自如下组的氨基酸的取代或缺失:
a)在位置1的丙氨酸,
b)在位置2的谷氨酸,
c)在位置3的丙氨酸,
d)在位置4的亮氨酸,
e)在位置5的亮氨酸,
f)在位置6的丙氨酸,
g)在位置7的亮氨酸,
h)在位置8的谷氨酰胺,
i)在位置9的谷氨酸,
j)在位置10的丙氨酸,
k)在位置11的丙氨酸,
l)在位置12的谷氨酸,
m)在位置13的天冬氨酸,
n)在位置14的苯丙氨酸,
o)在位置15的亮氨酸,
p)在位置16的缬氨酸,
q)在位置17的组氨酸,
r)在位置18的亮氨酸,
s)在位置19的苯丙氨酸,
t)在位置20的谷氨酸,
u)在位置21的天冬氨酸,
v)在位置22的丙氨酸,
w)在位置23的甲硫氨酸,
x)在位置24的亮氨酸,
y)在位置25的半胱氨酸,
z)在位置26的丙氨酸,
aa)在位置27的异亮氨酸,
bb)在位置28的组氨酸,及
cc)在位置29的丙氨酸。
在进一步特别优选的实施方案中,突变导致环1中确定的氨基酸的取代或缺失,其中在SEQ ID No.49的位置2的氨基酸天冬酰胺被取代,优选取代为缬氨酸,或者在SEQ IDNo.55的位置95的氨基酸丙氨酸被取代,优选取代为缬氨酸,或者在SEQ ID No.49的位置6的氨基酸脯氨酸被取代,优选取代为丝氨酸,或者在SEQ ID No.52的位置121的氨基酸脯氨酸被取代,优选取代为丝氨酸,或者在SEQ ID No.49的位置12的氨基酸色氨酸被取代,优选取代为终止信号,或者在SEQ ID No.52的位置127的氨基酸色氨酸被取代,优选取代为终止信号。
在进一步特别优选的实施方案中,突变导致在α2-螺旋中确定的氨基酸的取代或缺失,其中在SEQ ID No.1的位置1的氨基酸丙氨酸被取代,优选取代为苏氨酸,或者在SEQID No.58的位置107的氨基酸丙氨酸被取代,优选取代为苏氨酸,或者在SEQ ID No.1的位置4的氨基酸亮氨酸被取代,优选取代为苯丙氨酸、异亮氨酸或谷氨酰胺,或者在SEQ IDNo.52的位置132或者在SEQ ID No.34的位置92或者在SEQ ID No.38的位置130或者在SEQID No.61的位置106的氨基酸亮氨酸被取代,优选取代为苯丙氨酸、异亮氨酸或谷氨酰胺,或者在SEQ ID No.1的位置7的氨基酸亮氨酸被取代,优选取代为脯氨酸,或者在SEQ IDNo.61的位置109的氨基酸亮氨酸被取代,优选取代为脯氨酸,或者在SEQ ID No.1的位置8的氨基酸谷氨酰胺被取代,优选取代为终止信号或亮氨酸,或者在SEQ ID No.58的位置114或者在SEQ ID No.61的位置110的氨基酸谷氨酰胺被取代,优选取代为终止信号或亮氨酸,或者在SEQ ID No.1的位置10的氨基酸丙氨酸被取代,优选取代为苏氨酸,或者在SEQ IDNo.52的位置138的氨基酸丙氨酸被取代,优选取代为苏氨酸,或者在SEQ ID No.1的位置25的氨基酸半胱氨酸被取代,优选取代为酪氨酸,或者在SEQ ID No.52的位置153的氨基酸半胱氨酸被取代,优选取代为酪氨酸,或者在SEQ ID No.1的位置26的氨基酸丙氨酸被取代,优选取代为缬氨酸,或者在SEQ ID No.52的位置154的氨基酸丙氨酸被取代,优选取代为缬氨酸。
在本发明中,术语“突变”是指至少一个突变,优选一个突变,特别是唯一一个突变。在进一步优选的实施方案中,术语“至少一个突变”是指两个突变,特别是仅两个突变。在进一步优选的实施方案中,术语“至少一个突变”是指三个突变,特别是仅三个突变。在进一步优选的实施方案中,术语“至少一个突变”是指四个突变,特别是仅四个突变。在进一步优选的实施方案中,术语“至少一个突变”是指五个突变,特别是仅五个突变。在超过一个突变的情况中,突变也可以发生在不同的多核苷酸中,导致不同CENH3蛋白质(如果对于特定的植物物种存在的话)的CATD结构域中氨基酸序列改变。例如,大麦具有两个不同的CENH3蛋白质。
在本发明的一个优选实施方案中,突变是至少一个突变、至少两个突变、至少三个突变、至少四个突变,或者至少五个突变。
在本发明的一个优选实施方案中,最大突变数是2、3、4、5、6、7、8、9个,最优选10个。
在进一步优选的实施方案中,在CATD结构域中、优选在CATD结构域的环1或α2-螺旋中,存在一个氨基酸取代,特别是仅一个氨基酸取代。
在进一步优选的实施方案中,在CATD结构域中、优选在CATD结构域的环1或α2-螺旋中,存在两个氨基酸取代,特别是仅两个氨基酸取代。
在进一步优选的实施方案中,在CATD结构域中、优选在CATD结构域的环1或α2-螺旋中,存在三个氨基酸取代,特别是仅三个氨基酸取代。
在进一步优选的实施方案中,在CATD结构域中、优选在CATD结构域的环1或α2-螺旋中,存在四个氨基酸取代,特别是仅四个氨基酸取代。
在进一步优选的实施方案中,在CATD结构域中、优选在CATD结构域的环1或α2-螺旋中,存在五个氨基酸取代,特别是仅五个氨基酸取代。
在本发明的一个优选实施方案中,在CATD结构域中、优选在CATD结构域的环1或α2-螺旋中,存在1、1或2、1-3、1-4、1-5、优选1-6及更优选1-7个氨基酸取代。
特别地,本发明涉及在CENH3蛋白质内、特别是在其CATD结构域中导致或引起氨基酸取代的突变。因此,在本发明中,突变优选是在编码CENH3蛋白质的DNA序列中的非同义点突变或者取代,导致氨基酸改变。这也称作错义突变。进一步地,氨基酸改变或者氨基酸取代可以是保守的,即改变为具有相似物理化学性质的氨基酸;半保守的,例如负电荷氨基酸改变为正电荷氨基酸;或者是彻底改变,即改变为极为不同的氨基酸。
在本发明的一个优选实施方案中,具有单倍体诱导物生物活性的本发明的植物关于所述突变或者至少一个突变是纯合的。在本发明的进一步的实施方案中,具有单倍体诱导物生物活性的本发明的植物关于所述突变或者至少一个突变是杂合的。
本发明的植物具有单倍体诱导物生物活性。这是指在本发明的植物与野生型植物或表达野生型CENH3蛋白质的植物之间杂交时产生至少0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、优选至少1%、优选至少2%、优选至少3%、优选至少4%、优选至少5%、优选至少6%、优选至少7%、优选至少8%、优选至少9%、最优选至少10%、至少15%、至少20%或更多的单倍体后代。因此,野生型植物优选是相同物种的植物,其不包含根据本发明的植物在相应内源性CENH3基因内的突变,即植物能表达天然CENH3蛋白质,及表达野生型CENH3的植物优选是相同物种的植物,其包含i)无根据本发明的植物的突变的编码CENH3蛋白质的核苷酸序列,并能表达所述天然CENH3蛋白质,或者ii)编码来自另一植物物种的CENH3蛋白质的核苷酸序列,其示出与天然CENH3相当的功能性,例如来源于另一植物物种的这种CENH3蛋白质可以作为转基因导入。
因此,本发明最有利地提供了在广泛的真双子叶植物(eudicot)、双子叶植物和单子叶植物物种中产生单倍体诱导物株系的手段和方法。本发明还可以交换母本细胞质及在一个程序步骤中产生例如具有想要的基因型的细胞质雄性不育植物。在单一氨基酸突变可以通过诱变或任何其它非基于GMO的方法产生的范围内,本发明是有利的。
因此,在优选的实施方案中,通过应用单倍体诱导物株系单倍体化的完整程序的特征在于在至少一个位置具有本发明提供的改变的点突变的编码CENH3蛋白质的内源CENH3基因是非转基因的。
在本发明中,“内源”基因、等位基因或蛋白质是指植物的非重组序列,其与在各自的植物、特别是野生型植物中存在的序列相同。术语“突变的”是指人为改变的序列。举例的人为诱导的非转基因突变包括将植物暴露于高剂量化学、放射性或者其它诱变剂以选择突变体。或者,人为诱导的转基因突变,即重组改变或基因工程,例如利用TALE核酸酶、锌指核酸酶或者CRISPR/Cas***进行,包括DNA或氨基酸序列的融合、***、缺失和/或改变。
多核苷酸或多肽序列对于某生物体而言,如果其源自外来物种或者源自相同物种但是从其原来形式加以修饰,则其与所述生物体是“异源或外源的”。“重组”是指人为改变的,即转基因的多核苷酸或多肽序列。所用术语“转基因”为在本领域理解且是指通过对细胞基因组进行人为分子操纵如通过分子转化而将(优选异源的)核酸导入细胞中。因此,“转基因植物”是包含转基因的植物,即是经遗传修饰的植物。转基因植物可以是其中导入转基因的起始植物,其后代的基因组含有转基因。
术语“编码…的核苷酸序列”是指指导特定蛋白质,特别是CENH3蛋白质或其部分的表达的核酸。核苷酸序列包括被转录为RNA的DNA链序列及被翻译为蛋白质的RNA序列。核苷酸序列包括全长核酸序列以及衍生自全长序列的非全长序列。
术语“基因”是指编码核苷酸序列和相关的调节核苷酸序列、内含子、5’UTR和/或3’UTR。
术语“调节元件”是指位于核苷酸序列,优选编码序列(其转录和表达由调节元件控制,潜在地与细胞的蛋白质生物合成装置结合)的上游(5')、核苷酸序列内和/或下游(3')的序列,优选核苷酸序列。“调节”是指通过主要位于但不只是位于感兴趣的基因的转录起始处上游(5')的DNA序列元件诱导的基因表达的调节。调节可导致对于刺激全部应答或者无应答,或者其可以导致基因表达水平的变化。
如果两个序列的位置和朝向使得调节DNA序列影响编码DNA序列的表达,则调节元件,特别是DNA序列如启动子被称作“可操纵地连接于”编码RNA或蛋白质的DNA序列或者与其“关联”。
“启动子”是起始相关的DNA序列转录的DNA序列,特别是位于转录起始处上游(5’)及参与识别RNA聚合酶。根据特异性启动子区域,其也可以包含作为基因表达调节物的元件,如激活子、增强子和/或阻抑子。
“3'调节元件”(或者3'末端)是指确定正确的终止位点及含有聚腺苷酸化信号及能实现信使RNA(mRNA)加工或基因表达的任何其它调节信号基因部分,包含除了驱动转录起始的5'序列之外的DNA区段和基因的结构部分。聚腺苷酸化信号通常特征在于实现在mRNA前体的3'末端加入聚腺苷酸。通常通过存在与规范化形式5'-AATAAA-3'同源识别聚腺苷酸化信号。
术语“编码序列”是指编码蛋白质、多肽或其部分的基因部分,不包括驱动转录起始或终止的调节序列。
基因、编码序列或者调节元件可以是在细胞中正常发现的,在这种情况中称作“自体的”或者“内源的”,或者其可以是在细胞定位中不是正常发现的在这种情况中称作“异源的”、“转基因的”或者“转基因”。
“异源”基因、编码序列或者调节元件也可以细胞自体的,但是排列顺序和/或方向或者在基因组中位置或环境在其转移进的细胞中不是正常发现或者发生的。
术语“载体”是指重组DNA构建体,其可以是质粒、病毒、自主复制序列、人工染色体,如细菌人工染色体BAC、噬菌体或其它核苷酸序列,其中已经结合或重组了至少两个核苷酸序列,其中至少一个序列是本发明的核酸分子。载体可以是线性或环形的。载体可以由单链或双链DNA或RNA组成。
术语“表达”是指内源基因或转基因在植物中转录和/或翻译。
“转化”和“转移”是指将核酸分子,特别是DNA转移进细胞中的方法,包括但不限于基因枪方法如粒子轰击、显微注射、用各种物理方法例如电穿孔或者化学处理方法如聚乙二醇或PEG透过细胞膜;原生质体融合或者根癌农杆菌或发根农杆菌介导的转化。对于在植物细胞中注射或电穿孔DNA,对于使用的质粒没有特殊要求。可以使用质粒如pUC衍生物。如果从这种转化的细胞中再生完整植物,优选使用可选择标记。根据将想要的基因导入植物细胞中的方法,也许需要其它的DNA序列;如果例如Ti或Ri质粒用于植物细胞的转化,Ti和Ri质粒T-DNA的至少右侧边界、通常是右侧和左侧边界作为侧翼区与导入的基因连接。优选地,转移的核酸分子稳定地整合在受体植物的基因组或质体基因组中。
在本发明中,术语“单倍体诱导物生物活性”或者“单倍体诱导物”或者“单倍体诱导物株系”是指在与野生型植物或者至少表达野生型CENH3蛋白质的植物杂交时,在至少0.1%、至少0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、优选至少1%、优选至少2%、优选至少3%、优选至少4%、优选至少5%、优选至少6%、优选至少7%、优选至少8%、优选至少9%、最优选至少10%、最优选至少15%、最优选至少20%的情况中具有产生单倍体后代或子代能力的植物或植物株系。由于单倍体诱导物的染色体在减数***期间被消除,因此所得单倍体后代仅包含野生型亲本的染色体。然而,在单倍体诱导物是杂交的胚珠亲本的情况中,单倍体后代具有诱导物的细胞质及野生型亲本的染色体。
根据本发明,术语“植物”包括完整植物或者完整植物的部分。
完整植物优选是种子植物或者农作物。植物的部分是例如芽营养器官/结构,如叶、茎干和块茎;根,花和花器官(floral organ)/结构,例如苞片、萼片、花瓣、雄蕊、心皮、花药和胚珠;种子,包括胚、胚乳和种皮;果实和成熟子房;植物组织,例如维管组织、基本组织等;及细胞,如保卫细胞、***、毛状体等;以及其后代。
在任何情况中,本发明的植物包含至少一种细胞,所述细胞包含编码包含CATD结构域的着丝粒组蛋白H3蛋白质的核苷酸序列,其中所述核苷酸序列包含在CATD结构域中导致CENH3蛋白质的氨基酸序列改变的突变,所述改变可以赋予植物单倍体诱导物的生物活性,优选如在本文中更详细描述的。最优选地,本发明植物的大多数或者特别是所有细胞均包含如本文所述突变。
可用于本发明方法中的植物的物种优选是真双子叶植物、双子叶植物和单子叶植物。
在优选的实施方案中,术语“植物”只是涉及完整植物,即呈现出成熟植物的全部表型且能再生的植物、其发育早期阶段,例如植物胚,或者是指这二者。
在本发明的实施方案中,术语“植物”是指完整植物的一部分,特别是植物材料、植物细胞或植物细胞培养物。
术语“植物细胞”描述了植物的结构和生理学单位,其包含原生质体和细胞壁。植物细胞可以是分离的单细胞形式,如气孔保卫细胞或者培养的细胞,或者作为更高等组织单位(higher organized unit),例如植物组织或植物器官的部分。
术语“植物材料”包括植物的部分,特别是植物细胞、植物组织,特别是植物繁殖材料,优选叶、茎、根、萌发的(emerged)胚根、花或花的部分、花瓣、果实、花粉、花粉管、花药、花丝、胚珠、胚囊、***、子房、受精卵、胚、合子胚、体细胞胚、下胚轴切片、顶端分生组织、维管束、中柱鞘、种子、根、插枝、细胞或组织培养物,或者植物的任何其它部分或产物。
因此,本发明还提供了本发明植物的植物繁殖材料。所述“植物繁殖材料”应理解为是可以在体内或体外有性或无性繁殖的任何植物材料。在本发明范围内特别优选原生质体、细胞、愈伤组织、组织、器官、种子、胚、花粉、***、受精卵,以及得自转基因植物的任何其它繁殖材料。通过本发明的方法起源于突变的植物的植物部分如花、茎、果实、根或者先前突变、优选转化的及因此包含至少部分突变的细胞的其后代,也是本发明的一方面。
优选地,本发明的植物选自如下一组:大麦(大麦)、高粱(高粱)、黑麦(黑麦)、小黑麦、甘蔗(甘蔗)、玉米(玉米)、谷子(小米)、水稻(水稻)、小粒野生稻、澳洲野生稻、野生稻、小麦(小麦)、硬粒小麦(Triticum durum)、Hordeum bulbosum、紫色假雀麦(二穗短柄草)、海大麦(Hordeum marinum)、山羊草(山羊草)、苹果(苹果)、甜菜、向日葵(向日葵(Helianthus annuus))、澳大利亚胡萝卜(Daucus glochidiatus)、美国野胡萝卜(Daucuspusillus)、Daucus muricatus、胡萝卜(胡萝卜)、桉树(巨桉)、Erythranthe guttata、Genlisea aurea、林地烟草(美花烟草)、烟草(烟草)、绒毛烟草、番茄(番茄)、马铃薯(马铃薯)、咖啡(中果咖啡)、葡萄(葡萄)、黄瓜(黄瓜)、桑树(桑树)、拟南芥(thale cress)(拟南芥)、深山南芥、沙石水芹(sand rock-cress)(Arabidopsis arenosa)、须弥芥、卵叶须弥芥、波状野荠菜(弯曲碎米荠)、胡椒草(北美独行菜)、荠菜(荠菜)、Olmarabidopsispumila、毛状石水芹(hairy rockcress)(硬毛南芥)、油菜(甘蓝型油菜)、花椰菜(甘蓝)、芜菁、Brassica juncacea、黑芥(黑芥菜)、萝卜(萝卜)、芝麻菜苜蓿、橙(甜橙)、麻风树、大豆、以及黑三角叶杨(毛果杨)。
特别优选所述植物选自如下一组:大麦(大麦)、高粱(高粱)、黑麦(黑麦)、小黑麦、甘蔗(甘蔗)、玉米(玉米)、水稻(水稻)、小麦(小麦)、硬粒小麦、燕麦(Avena sativa)、Hordeum bulbosum、甜菜、向日葵(向日葵)、胡萝卜(胡萝卜)、烟草(烟草)、番茄(番茄)、马铃薯(马铃薯)、咖啡(中果咖啡)、葡萄(葡萄)、黄瓜(黄瓜)、拟南芥(拟南芥)、油菜(甘蓝型油菜)、花椰菜(甘蓝)、芜菁、Brassica juncacea、黑芥(黑芥菜)、萝卜(萝卜)和大豆。
在优选的实施方案中,本发明的植物含有作为内源基因或者转基因的的编码CENH3的核苷酸序列。
在优选的实施方案中,本发明涉及根据本发明教导的植物,其中在编码CENH3的核苷酸序列中经非转基因或转基因地导入所述改变。
因此,优选在实施方案中,其中所述突变是在内源CENH3基因中产生,获得的植物是非转基因的。优选地,所述突变是通过非转基因诱变、特别是化学诱变实现的,优选通过EMS(甲基磺酸乙酯)诱导的TILLING或者靶向基因组编辑方法实现。
因此,本发明涉及一种植物,其中非转基因导入突变是通过化学诱变、特别是通过TILLING实现的,所述突变在CATD结构域中导致CENH3蛋白质的氨基酸序列改变,优选氨基酸取代或缺失,及所述改变赋予植物单倍体诱导物生物活性。
在另一个优选的实施方案中,所述突变以转基因形式导入植物中。优选地,这是通过转化包含编码包含改变的CENH3的至少CATD结构域的核苷酸序列的载体实现的,优选如本文所述。转化植物及将转基因导入植物基因组中的方法为本领域熟知。
因此,在优选的实施方案中提供了一种植物,其中在CENH3蛋白质内经转基因导入改变,优选氨基酸取代或氨基酸缺失是通过载体转化实现的,所述载体包含这样的核苷酸序列,其编码位于CATD结构域中至少环1及对应于来源于拟南芥的SEQ ID No.38所示CENH3蛋白质的位置340-378的核苷酸,但是包含SEQ ID No.49所示共有序列或者表1所示的确定的氨基酸的至少一个氨基酸取代或缺失,或者编码位于CATD结构域中的至少α2-螺旋及对应于衍生自拟南芥的SEQ ID No.38所示CENH3蛋白质的位置379-465的核苷酸,但是包含SEQ ID No.1所示共有序列或表2所示的确定的氨基酸的至少一个氨基酸取代。在另一个实施方案中提供了一种植物,其中在CENH3蛋白质内导入氨基酸取代或缺失是通过载体转化实现的,所述载体包含这样的核苷酸序列,其编码至少CATD结构域或者包含CATD结构域的CENH3蛋白质,包含在SEQ ID No.49或1所示共有序列或表1或2所示的确定的氨基酸的至少一个氨基酸取代或缺失。
优选地,农杆菌介导的转化、蘸花法或者粒子轰击法用于进行转化。
在优选的实施方案中,其中编码根据本发明的突变的CENH3蛋白质的核苷酸序列以转基因形式被转化进植物中,及内源基因的一或两个等位基因优选被失活或敲除。在另一优选的实施方案中,其中编码根据本发明的突变的CENH3蛋白质的核苷酸序列以转基因形式被转化进植物中,所述转基因是过表达的,以作为更有竞争力的内源CENH3蛋白质优选在着丝点复合物产生期间更有竞争力。
本发明还提供了通过本发明的方法可获得、特别是获得的植物,其特征在于具有单倍体诱导物生物活性。
在本发明的优选实施方案中,产生根据本发明的具有单倍体诱导物生物活性的植物的方法本质上不是生物学方法。
进一步地,本发明还提供了产生根据本发明的具有单倍体生物活性的植物的方法,包括如下步骤:
i)将植物的种子暴露于足量的诱变剂甲基磺酸乙酯(EMS)以获得M1植物,
ii)使得可育的M2植物足量产生,
iii)分离M2植物的基因组DNA,及
iv)选择具有导致CENH3的CATD结构域中氨基酸序列改变的突变的个体。
在优选的实施方案中,本发明进一步涉及一种产生根据本发明的具有单倍体诱导物生物活性的植物的方法,包括如下步骤:
xx)提供包含编码CENH3蛋白质的至少CATD结构域的核苷酸序列的载体,其包含导致在CATD结构域中CENH3蛋白质的氨基酸序列改变的突变,
yy)用所述载体转化植物细胞,其中优选包含CENH3基因的一或两个内源等位基因失活或敲除的植物细胞,及
zz)从所述植物细胞再生具有单倍体诱导物生物活性的植物。
在优选的实施方案中,本发明进一步涉及一种产生根据本发明的具有单倍体诱导物生物活性的植物的方法,包括如下步骤:
yy)用编码CENH3蛋白质的至少CATD结构域的核苷酸序列转化植物细胞,其包含导致在CATD结构域中CENH3蛋白质的氨基酸序列改变的突变,或者用包含编码CENH3蛋白质的至少CATD结构域的核苷酸序列的载体转化植物细胞,所述核苷酸序列包含导致在CATD结构域中CENH3蛋白质的氨基酸序列改变的突变,及
zz)从所述植物细胞中再生具有单倍体诱导物生物活性的植物。
特别地,本发明涉及通过如下方法可获得、特别是获得的单倍体植物:
a)将根据本发明的具有单倍体诱导物生物活性的植物与表达野生型CENH3蛋白质的植物杂交,任选地,
b)鉴别从所述杂交步骤中产生的单倍体后代。
优选地,鉴别的单倍体植物可以转变为双单倍体植物,优选通过秋水仙素处理转变,这也是本发明的一部分。因此,本发明还涉及将根据本发明的单倍体植物转变为双单倍体植物可获得、特别是获得的双单倍体植物,优选通过秋水仙素处理或者通过染色体自然加倍方法转变。
因此,本发明还提供了一种产生单倍体植物的方法,包括如下步骤:
a)将根据本发明的具有单倍体诱导物生物活性的植物与表达野生型CENH3蛋白质的植物杂交,及
b)鉴别从所述杂交步骤中产生的单倍体后代。
在进一步的步骤c)中,将所选择的单倍体植物转变为双单倍体植物,优选通过秋水仙素处理转变。因此,本发明还涉及一种产生双单倍体植物的方法。
在本发明的优选实施方案中,本发明提供的方法本质上不是生物学方法。
特别地,本发明方法不是仅依赖于自然现象如杂交或选择,特别是不是由自然现象如杂交或选择组成,事实上是基本基于这样的技术教导以提供通过人为制备的特异性突变的核苷酸序列。因此,本发明在本发明的核苷酸序列和植物中导入称作突变的一种特定结构特征,该突变不是由于任何自然现象如杂交或选择导致或与其相关。
在本发明的具体实施方案中,提供了一种包括杂交步骤的方法,所述杂交步骤不提供-如通常的杂交提供的-杂合后代,事实上提供纯合后代。进一步地,后代的单倍性不是用于有性杂交的植物的的基因混合所致。此外,在自然界找不到本发明权利要求的产生双单倍体植物的方法。
进一步地,本发明还提供了一种促进细胞质交换的方法,包括如下步骤:
x)将本发明的植物作为胚珠亲本与作为花粉亲本的表达野生型CENH3蛋白质的植物杂交,及
y)获得包含花粉亲本染色体和胚珠亲本细胞质的单倍体后代。
在本发明的优选实施方案中,本发明提供的方法本质上不是生物学方法。所述方法由于上述相同原因而本质上不是生物学方法,特别是由于其不是由自然现象如杂交和选择完全组成,而是包含一种显著的技术教导的基本特征,由此提供在本发明的核苷酸序列和植物中特定的突变。此外,后代的单倍性不是用于有性杂交的植物基因混合所致。
所述方法可有利地用于产生细胞质雄性不育(CMS)。CMS是由于细胞核外基因组(线粒体或叶绿体)导致,并示出母系遗传性。因此,本发明的植物呈现出CMS,且是杂交的胚珠亲本。以此方式,CMS可以导入杂交配体中,优选是作物精英株系。
在优选的实施方案中,本发明的植物可以用于恢复雄性生育力的方法,为CMS的杂交亲本提供正常的细胞质。通过这种杂交,CMS亲本的染色体导入非CMS的本发明单倍体诱导物的正常细胞质中。然而,CMS植物的花粉产生需要通过温度、光照、日照长度等诱导。
不受理论限制,在图中示出了本发明的方法、特别是单亲染色体消除方法怎样在诱导物CENH3x野生型CENH3种间杂交胚中工作的模型。(A)可能单倍体诱导物来源的***含有较少的CENH3或者与野生型相比降低的未知“CENH3-跨世代需要的特征(transgeneration required signature)”。母本CENH3的量减少不太可能,因为根据使用CENH3-GFP报告蛋白在拟南芥植物中进行的研究,由CENH3标记的是***核而不是***。然而,仍可能的是产生“着丝粒印迹”的剩余的母本CENH3传递至后代。(B)在受精后几小时内,将父本野生型CENH3从受精卵核中主动除去,及(C)在拟南芥中在胚乳发育的16核阶段发生合子中的CENH3-GFP的着丝粒再荷载。(D)在经历单倍体化的胚中,母本染色体的着丝粒再荷载被削弱或延迟,由于在细胞***后期期间着丝粒失活导致滞后的染色体。随后,微核化的单倍体诱导物染色体降解及(E)单倍体胚产生。单倍体胚在母本细胞质背景中含有得自父本的染色体。
本发明还涉及编码CENH3蛋白质的至少CATD结构域或者包括结构域的蛋白质的核苷酸序列或者包括包含导致CATD结构域中CENH3蛋白质的氨基酸序列改变的突变的CATD结构域的CENH3蛋白质。
本发明还涉及包含所述核苷酸序列及如果存在的话优选如本文所示结合序列的载体特别是病毒载体、构建体或质粒载体。
在本发明特别优选的实施方案中,编码CENH3蛋白质的至少CATD结构域的核苷酸序列包含至少CENH3的完整编码区,特别是CENH3的基因。
在本发明进一步优选的实施方案中,CENH3的编码序列可以与调节元件如5'和/或3'调节元件结合,最优选与启动子、优选组成型或可诱导启动子结合。
此外,本发明提供了包含所述核苷酸序列的植物细胞或包含所述核苷酸序列作为转基因的载体。
在本发明中,如本文所用术语“包含”是指具有“包括”或“含有”的含义,是指除了明确提及的要素之外也可能存在其它要素。
在本发明的优选实施方案中,如本文所用术语“包含”也应理解为是指“由…组成”,从而不包括除了明确提及的要素之外的其它要素。
在进一步优选的实施方案中,如本文所用术语“包含”也应理解为是指“基本由…组成”,从而不包括除了明确提及的要素之外显著有助于公开的教导的其它要素。
本发明进一步优选的实施方案是在权利要求书中的主题。
本发明通过如下非限制性实施例和两个图表得以更详细阐述。
序列方案示出:
SEQ ID No.1:CENH3α2-螺旋的共有氨基酸序列,
SEQ ID No.2-32:用于本发明中的引物的核苷酸序列,
SEQ ID No.33:大麦的野生型β-CENH3蛋白质的cDNA核苷酸序列,
SEQ ID No.34:大麦的β-CENH3的氨基酸序列,
SEQ ID No.35:大麦(TILLING株系4528突变体)的β-CENH3的cDNA序列,
SEQ ID No.36:大麦(TILLING株系4528突变体)的β-CENH3的氨基酸序列,
SEQ ID No.37:拟南芥CENH3的野生型编码序列(cDNA)的核苷酸序列,
SEQ ID No.38:野生型拟南芥CENH3氨基酸序列,
SEQ ID No.39:突变的拟南芥CENH3(突变体L-I)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.40:突变的拟南芥CENH3(突变体L-I)的氨基酸序列,
SEQ ID No.41:突变的拟南芥CENH3(突变体L-F)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.42:突变的拟南芥CENH3(突变体L-F)的氨基酸序列,
SEQ ID No.43:甜菜CENH3的野生型编码序列(cDNA)的核苷酸序列,
SEQ ID No.44:野生型甜菜CENH3的氨基酸序列,
SEQ ID No.45:甜菜CENH3(突变体L-F)的编码序列的核苷酸序列,
SEQ ID No.46:突变的甜菜CENH3(突变体L-F)的氨基酸序列,
SEQ ID No.47:甜菜CENH3(突变体L-I)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.48:突变的甜菜CENH3(突变体L-I)的氨基酸序列,
SEQ ID No.49:CENH3环1的共有氨基酸序列,
SEQ ID No.50:甘蓝型油菜CENH3的野生型基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.51:甘蓝型油菜CENH3的野生型编码序列(cDNA)的核苷酸序列,
SEQ ID No.52:野生型甘蓝型油菜CENH3的氨基酸序列,
SEQ ID No.53:高粱CENH3的野生型基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.54:高粱CENH3的野生型编码序列(cDNA)的核苷酸序列,
SEQ ID No.55:野生型高粱CENH3的氨基酸序列,
SEQ ID No.56:玉米CENH3的野生型基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.57:玉米CENH3的野生型编码序列(cDNA)的核苷酸序列,
SEQ ID No.58:野生型玉米CENH3的氨基酸序列,
SEQ ID No.59:甜菜CENH3野生型基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.60:甜菜CENH3的野生型编码序列(cDNA)的核苷酸序列,
SEQ ID No.61:野生型甜菜CENH3的氨基酸序列,
SEQ ID No.62:突变的甘蓝型油菜CENH3(突变体P121S)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.63:突变的甘蓝型油菜CENH3(突变体P121S)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.64:突变的甘蓝型油菜CENH3(突变体P121S)的氨基酸序列,
SEQ ID No.65:突变的甘蓝型油菜CENH3(突变体W127stop)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.66:突变的甘蓝型油菜CENH3(突变体W127stop)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.67:突变的甘蓝型油菜CENH3(突变体W127stop)的氨基酸序列,
SEQ ID No.68:突变的甘蓝型油菜CENH3(突变体L132F)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.69:突变的甘蓝型油菜CENH3(突变体L132F)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.70:突变的甘蓝型油菜CENH3(突变体L132F)的氨基酸序列,
SEQ ID No.71:突变的甘蓝型油菜CENH3(突变体A138T)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.72:突变的甘蓝型油菜CENH3(突变体A138T)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.73:突变的甘蓝型油菜CENH3(突变体A138T)的氨基酸序列,
SEQ ID No.74:突变的甘蓝型油菜CENH3(突变体C153Y)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.75:突变的甘蓝型油菜CENH3(突变体C153Y)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.76:突变的甘蓝型油菜CENH3(突变体C153Y)的氨基酸序列,
SEQ ID No.77:突变的甘蓝型油菜CENH3(突变体A154V)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.78:突变的甘蓝型油菜CENH3(突变体A154V)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.79:突变的甘蓝型油菜CENH3(突变体A154V)的氨基酸序列,
SEQ ID No.80:突变的玉米CENH3(突变体A107T)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.81:突变的玉米CENH3(突变体A107T)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.82:突变的玉米CENH3(突变体A107T)的氨基酸序列,
SEQ ID No.83:突变的玉米CENH3(突变体Q114stop)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.84:突变的玉米CENH3(突变体Q114stop)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.85:突变的玉米CENH3(突变体Q114stop)的氨基酸序列,
SEQ ID No.86:突变的高粱CENH3(突变体A95V)的基因组序列(基因组DNA)的核苷序列,
SEQ ID No.87:突变的高粱CENH3(突变体A95V)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.88:突变的高粱CENH3(突变体A95V)的氨基酸序列,
SEQ ID No.89:突变的甜菜CENH3(突变体L106Q)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.90:突变的甜菜CENH3(突变体L106Q)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.91:突变的甜菜CENH3(突变体L106Q)的氨基酸序列,
SEQ ID No.92:突变的甜菜CENH3(突变体L109P)的基因组序列的核苷酸序列,
SEQ ID No.93:突变的甜菜CENH3(突变体L109P)的编码序列(cDNA)的核苷酸序列,
SEQ ID No.94:突变的甜菜CENH3(突变体L109P)的氨基酸序列,
SEQ ID No.95:突变的甜菜CENH3(突变体Q110L)的基因组序列(基因组DNA)的核苷酸序列,
SEQ ID No.96:突变的甜菜CENH3(突变体Q110L)的编码序列(cDNA)的核苷酸序列,及
SEQ ID No.97:突变的甜菜CENH3(突变体Q110L)的氨基酸序列。
图1示出关于本发明方法的机理模型。
图2示出拟南芥(第一行)、甜菜(第二行)、甘蓝型油菜(第三行)、玉米(第四行)、高粱(第五行)的氨基酸序列的比对以及示出这五个植物物种的保守水平的图表。
实施例
实施例1:通过靶向基因组中局部损伤(TILLING)诱变大麦α和βCENH3
为了鉴别内源CENH3的单一点突变是否可以产生单倍体诱导物,筛选二倍体大麦(Hordeum vulgare)的EMS诱导的TILLING种群(Gottwald et al.,2(2009),BMC ResNotes,258),这是编码两个CENH3的有功能的变体(α和βCENH3)的物种(Sanei et al.,108(2011),Proc Natl Acad Sci U S A,E498-505)。假设任一CENH3变体互补,αCENH3或βCENH3的功能性突变仍可以产生子代。
为此,筛选cv.Barke的10,279株经EMS处理的二倍体大麦(Hordeum vulgare)植物的TILLING种群以鉴别α和βCENH3突变体等位基因。用如下四个和三个引物组合分别扩增α和βCENH3变体的所有外显子及部分相应内含子,使用如先前描述的异源双链步骤进行PCR(Gottwald et al.,(2009),BMC Res Notes,258):
Hv_aCENH3_EX1+2+3_F:AGGCAGGGTCTCAATTCCTT(SEQ ID No.2),
Hv_aCENH3_EX1+2+3_R:GTCCCATCATCCATCGTCTT(SEQ ID No.3),
Hv_aCENH3_EX4+5_F:CCCACTTCCTTGTTGTGGAC(SEQ ID No.4),
Hv_aCENH3_EX4+5_R:GGCGATAAATGTATCTTGCATTC(SEQ ID No.5),
Hv_aCENH3_EX6_F:TGGTAGCAACCAGAGCTACG(SEQ ID No.6),
Hv_aCENH3_EX6_R:ACTGGCATGTTTCCTTCTGC(SEQ ID No.7),
Hv_aCENH3_EX7_F:CGGACGGAGGGAGTATTTCT(SEQ ID No.8),
Hv_aCENH3_EX7_R:GGACATGCCCAAAGAAAGTG(SEQ ID No.9),
Hv_bCENH3_EX1+2_F:GCCAGCGAGTACTCCTACAAG(SEQ ID No.10),
Hv_bCENH3_EX1_R:TTGAGTTACCAGCCACCACTC(SEQ ID No.11),
Hv_bCENH3_EX3_F:GTCATGCACTGTGTCTTGCA(SEQ ID No.12),
Hv_bCENH3_EX3_R:TGCTAAGATCGGATAACTGTGG(SEQ ID No.13),
Hv_bCENH3_EX4_F:TGCTCCTGAACAAACTGAACC(SEQ ID No.14),
Hv_bCENH3_EX4_R:GTGGCCGTCAGTACAATCG(SEQ ID No.15)
PCR产物用dsDNA裂解试剂盒消化并使用Mutation Discovery Kit和Gel-dsDNA试剂试剂盒在AdvanCETM FS96***上根据厂商指导(Advanced Analytical,IA,USA)进行分析。
RNA提取、PCR和定量实时RT-PCR
使用Trizol方法(Chomczynski and Sacchi,162(1987),Anal Biochem,156-159)从根、叶中分离总RNA,根据厂商指导通过Picopure RNA分离试剂盒(Arcturus)从花药(在减数***和成熟花粉发育之间的显微镜下分期)、心皮、胚乳和胚中分离。使用GAPDH引物(见表3)通过PCR证实不存在基因组DNA污染。10μl的PCR混合物含有1μl的cDNA模板、5μl的2×Power SYBR Green PCR Master Mix(Applied Biosystems)、0.33mM的每个基因的正向和反向引物(见表3)。反应在Applied Biosystems 7900HT Fast Real-Time PCR***中进行。使用如下条件进行PCR:95℃进行10分钟,随后是在95℃进行15秒、在60℃退火温度进行60秒的40次循环。对于每个cDNA样品进行三次技术复制。快速实时PCR***和数据使用SDS软件v 2.2.2分析。将每个基因的转录水平根据如下公式标准化为GAPDH:R=2^(-(CtGOI–CtH))*100,其中R=相对改变,GOI=感兴趣的基因,H=管家(GAPDH)。这两种引物的特异性和效力使用克隆的全长大麦α和βCENH3基因的系列稀释的质粒通过qRT-PCR确定。等量质粒的相似Ct值(在报道蛋白染料的荧光信号超过背景水平的PCR循环)表明这两种引物均可以相同效力扩增特异性转录物。
表3
引物名称 | 序列(5’to 3’) |
GAPDH-F | CAATGATAGCTGCACCACCAACTG(SEQ ID No.21) |
GAPDH-R | CTAGCTGCCCTTCCACCTCTCCA(SEQ ID No.22) |
Hvα-F | AGTCGGTCAATTTTCTCATCCC(SEQ ID No.23) |
Hvα-R | CTCTGTAGCCTCTTGAACTGC(SEQ ID No.24) |
Hvβ-F | GCCATTGTCGAACAAGAAGG(SEQ ID No.25) |
Hvβ-R | TAACACGGTGCGAATGAATG(SEQ ID No.26) |
CH3A+L130_F_for | phos-GACAGCTGAAGCATTTGTTGCTCTTC(SEQ ID No.27) |
CENH3L130_I_for | phos-GACAGCTGAAGCTATTGTTGCTCTTC(SEQ ID No.28) |
CENH3L130_F+I_rev | phos-CAACGATTGATTTGGGGAGGG(SEQ ID No.29) |
cenh3-1_mut_for | GGTGCGATTTCTCCAGCAGTAAAAATC(SEQ ID No.30) |
cenh3-1_mut_rev | CTGAGAAGATGAAGCACCGGCGATAT(SEQ ID No.31) |
cenh3-1_mut2429r | AACTTTTGCCATCCTCGTTTCTGTT(SEQ ID No.32) |
对于两种大麦CENH3变体仅鉴别了错义点突变。
使用CENH3变体特异性抗体通过对着丝粒进行免疫染色检测纯合M2突变体的突变的CENH3s的非功能性。对大麦野生型和纯合的TILLING株系4528(根据本发明的植物)的有丝***和减数***染色体使用特异于αCENH3和βCENH3的抗体进行免疫染色。在着丝粒的αCENH3和βCENH3信号在所有突变体中均显示,仅在氨基酸位置92(SEQ ID No.36)、即在SEQID No.1所示共有序列氨基酸位置4的亮氨酸取代为苯丙氨酸的纯合的TILLING株系4528,显示为在有丝***、减数***和间期细胞中无着丝粒βCENH3信号。在大麦β-CENH3序列的SEQ ID No.36所示氨基酸位置92的亮氨酸取代为苯丙氨酸对应于在大麦β-CENH3cDNA序列(SEQ ID No.35)位置274的C取代为T的单一核苷酸取代。
在整个株系中发现在着丝粒外的仅微弱分散的βCENH3信号。测量α和βCENH3在野生型(cv Barke)(SEQ ID no.33和34)及在具有突变的βCENH3的TILLING株系4528中的转录水平。在不同组织中使用特异性引物(表3)测量α和βCENH3的相对表达水平。从总RNA制备cDNA,并将基因表达水平标准化为磷酸甘油脱氢酶(GRPTA)的表达水平。显然,突变的βCENH3变体的着丝粒荷载似乎受损,而发现野生型与突变的βCENH3之间的转录水平无差异。因此,只由αCENH3组成的着丝粒在大麦中足以发挥有丝***着丝粒功能,因为发现没有明显的染色体分离缺陷如后期桥以及倍性或周期值改变。此外,在突变体植物中观测到植物***、周期值和生长表型中检测不到显著差异。
需要解决失去的βCENH3是否由额外αCENH3补偿以维持突变体中着丝粒的功能的这个问题。因此,通过像素强度测量对野生型(测量126个着丝粒)和株系4528(测量56个着丝粒)的αCENH3免疫染色信号进行量化对比。突变体中αCENH3增加19.8%表明失去的βCENH3通过额外掺入αCENH3而部分补偿。βCENH3突变位于由组蛋白折叠的部分α1螺旋、环1和α2螺旋限定的进化高保守的靶向结构域(CATD)。这个结构域是通过伴侣蛋白经着丝粒荷载CENH3所需要的。
间接免疫染色
如先前所述进行核及染色体的间接免疫染色(Sanei et al.,108(2011),ProcNatl Acad Sci USA,E498-505)。使用豚鼠抗-αCENH3-特异性和兔抗-βCENH3-特异性抗体检测大麦的CENH3。兔HTR12-特异性抗体(abcam,ab72001)用于检测拟南芥CENH3(AtCENH3)。用制冷的(cooled)CCD-相机(ORCA-ER,Hamamatsu)记录荧光显微镜信号。通过使用Olympus BX61显微镜和ORCA-ER CCD相机(Hamamatsu)进行成像。为了分析免疫信号和核染色质的结构,应用在光学分辨率为~100nm(超级分辨率)的Structured IlluminationMicroscopy(SIM),使用Elyra显微镜***的C-Apo 63x/1.2W Korr物镜和软件ZEN(Zeiss,Germany)进行。使用合适的激发和发射滤器,对于每个荧光物单独捕获图像。将图像使用Adobe Photoshop 6.0软件合并。为了确定核中α和βCENH3荧光强度中的量,使用TINA 2.0软件通过ZEN软件从样本的光学SIM截面堆栈中产生的以最大强度投影测量荧光强度。设定强度阈值以从图片中计算性减去背景像素。在核内的所有信号的灰度值的校正的总和用于确定CENH3含量。使用ZEN软件基于SIM图片堆叠进行3D渲染。
实施例2:拟南芥
为了论证CATD结构域中的突变导致观测到受损的着丝粒荷载,将eYFP与拟南芥CENH3(SEQ ID No.37,蛋白质SEQ ID No.38)的编码序列(CDS)在N末端融合,拟南芥CENH3中具有L/I(亮氨酸/异亮氨酸)(CDS:SEQ ID No.39,蛋白质:SEQ ID No.40)或者L/F(亮氨酸/苯丙氨酸)(CDS:SEQ ID No.41,蛋白质:SEQ ID No.42)相应位置交换(L130I或L130F,对应于在大麦的βCENH3氨基酸位置92,即SEQ ID No.1所示共有序列的氨基酸位置4)。在拟南芥的位置130的亮氨酸取代为异亮氨酸对应于在SEQ ID No.37的位置388的C取代为A的单一核苷酸取代。
在位置130的氨基酸亮氨酸取代为苯丙氨酸是由于在SEQ ID No.37的位置387和388的两个核苷酸取代即TC取代为AT所致。
用相应的抗野生型CENH3和抗GFP的双重标记转基因拟南芥显示,突变的CENH3的着丝粒靶向显著降低。
接着,为了检测单倍体诱导物能力,将具有L130I或L130F交换的拟南芥基因组CENH3构建体用于转化杂合的CENH3敲除拟南芥植物(Ravi and Chan,Nature,464(2010),615-618)。基因型分型鉴别纯合CENH3无效突变体,其由基因组CENH3野生型、L130I或L130F构建体补足。由于获得含有任一构建体的存活的二倍体植物,很可能这个突变在纯合的拟南芥植物中不削弱着丝粒功能。当将表达点突变的L130F CENH3蛋白质的CENH3无效突变体与野生型杂交时,来自突变体的染色质被消除,产生单倍体后代。流式细胞分析证实10.7%的F1植物是单倍体。
CENH3转基因的克隆与产生
为了产生具有导致苯丙氨酸130(F130)和异亮氨酸130(I130)代替野生型亮氨酸130(L130)的突变的CENH3基因组片段,将用于补足cenh3-1/cenh3-1(cenh3无效突变体)的pCAMBIA1300载体中基因组CENH3片段(Ravi and Chan,Nature,464(2010),615-618;Raviet al.,Genetics,186(2010),461-471)通过唯一的HindIII和BamHI位点亚克隆进pBlueScript II KS(Strategene,www.stratagene.com)。在pBlueScript II KS中使用定向诱变试剂盒(Finnzymes,www.finnzymes.com)根据厂商指导略加改变产生CENH3的突变L130I或L130F。下列5'-磷酸化的引物用于诱变:CH3A+L130_F_for、CENH3L130_I_for和CENH3L130_F+I_rev。将突变的CENH3基因组片段通过唯一的HindIII和BamHI位点亚克隆进含有潮霉素抗性标记的初始pCAMBIA1300中。所有构建体通过测序核实。引物见上表3。
为了产生含有在CENH3CDS内导致L130I或L130F的突变的p35S::eYFP-CENH3融合构建体,将含有p35S::eYFP-CENH3表达(Lermontova,18(2006),Plant Cell,615-618)的质粒(p35S-BM,Schmidt,www.dna-cloning.com)用作定向诱变试剂盒(Finnzymes,www.finnzymes.com)的模板。导入希望的突变的引物和策略与上述相同。将获得的表达盒(35Spro,eYFP-(突变的)CENH3和NOS终止子)通过唯一的SfiI限制位点亚克隆进含有草丁膦抗性标记的pLH7000载体中(Schmidt,www.dna-cloning.com),并通过测序核实。
植物转化、培养条件和异花授粉
将拟南芥A.thaliana野生型(SEQ ID No.37和38)及cenh3-1/CENH3杂合子植物(均登记为Columbia-0)通过蘸花法转化(Clough and Bent,16(1998),Plant J,735-743)。在含有相应抗生素的Murashige和Skoog固体培养基上选择转基因后代。使植物在培养皿上在长日照条件(20℃16h光照/18℃8h黑暗)下发芽,在短日照条件(20℃8h日照/18℃16h黑暗)下生长4周,然后再移至长日照条件下。对于杂交,将突变的cenh3A.thaliana的闭合的花蕾通过用镊子除去未成熟的花药而去雄。去雄的花蕾的柱头用来自新开放的野生型拟南芥花的成熟花药的淡黄色花粉授粉。
拟南芥的DNA提取及基因型分型
使用标准方法进行基因组DNA制备和基于PCR的基因型分型。根据Edwards et al.(1991),Nucleic Acids Res 19,1349所述提取DNA。
在dCAPS基因型分型反应中对植物进行cenh3-1的基因型分型。使用dCAPS引物cenh3-1_mut_for和cenh3-1_mut_rev扩增CENH3。扩增子用EcoRV消化,并在凝胶上分辨(resolved)。cenh3-1突变体等位基因未切割(215bp),而WT CENH3等位基因被切割(191和24bp)。引物见表3。为了在用CENH3基因组基因座(L130、L130I或L130F未标记的CENH3转基因)转化的cenh3-1/CENH3植物子代中对内源CENH3基因座进行cenh3-1基因型分型,使用转基因CENH3基因座之外的引物进行初始PCR反应,使得内源的而不是转基因的CENH3基因座特异性扩增。使用的引物是cenh3-1_mut_for和cenh3-1_mut2320r/cenh3-1_mut2429r。纯化扩增子并用作如上述cenh3-1植物的第二个dCAPS PCR基因型分型反应的模板。引物见表3。通过PCR核实转基因的存在。
植物和种子的流式细胞分析
对于植物的流式细胞倍性分析,在补加了DNas-free RNase(50μg/ml)和碘化丙啶(50μg/ml)核分离缓冲液中(Galbraith et al.(1983),Science 220,1049-1051)用锋利刀片将5-10个个体的等量叶材料同时割下。将该核悬浮液通过35μm细胞过滤器盖过滤进5ml聚苯乙烯管中(BD Biosciences),并在装备了氩离子激光器INNOVA 90C(Coherent)的FACStarPLUS细胞分选仪(BD Biosciences)上测量。测量大约10,000个核,并使用软件CELLQuest ver.3.3(BD Biosciences)分析。所得柱状图与代表二倍体野生型植物的参考柱状图对比。在检测到在单倍***置出现另外的峰的情况中,将植物单独再次测量以鉴定单倍体个体。
如上所述使用核分离缓冲液MA VI(100mM Tris-HCl,5.3mM MgCl2,86mM NaCl,30.6mM柠檬酸钠,1.45mM Triton X-100,pH 7.0;补加50μg/ml DNas-free RNase和50μg/ml碘化丙啶)进行种子的核分离。核悬浮液在FACSAria细胞分选仪(BD Biosciences)上测量,并使用FACS Diva软件ver.6.1.3(BD Biosciences)分析。与上文所述相似,集合前10-20个种子以鉴别具有单倍体胚的株系,及在第二步中将单一的种子与萝卜(GenebankGatersleben,登记号RA 34)的叶材料一起切碎,作为内部参考以证实单倍体种子的存在。
实施例3:甜菜
进一步地,也在甜菜中测定鉴别的突变的功能意义。产生RFP报道蛋白构建体,其含有甜菜CENH3的cDNA(SEQ ID No.43,蛋白质SEQ ID No.44),具有L106F(SEQ ID No.45,蛋白质SEQ ID No.46)或者L106I(SEQ ID No.47,蛋白质SEQ ID No.48)交换(对应于大麦的氨基酸位置92,SEQ ID No.1所示共有序列的氨基酸位置4),并用于甜菜的稳定转化及检测突变的CENH3的降低的着丝粒靶向。
在位置106的亮氨酸取代为苯丙氨酸的氨基酸取代是由于两个核苷酸取代所致,即在SEQ ID no.43的位置316的C取代为T及在位置318的G取代为T。
在位置106的亮氨酸取代为异亮氨酸的氨基酸取代是由于两个核苷酸取代所致,即在SEQ ID no.43的位置316的C取代为A及在位置318G取代为T。
植物转化与培养条件
将6-8周龄甜菜野生型植物叶(在半控温室条件下生长)通过微粒轰击(用0.5μg质粒DNA包被的300μg金微粒)进行瞬时转化。将经轰击的叶保温48-72小时(25℃,16h光照(350μmolm-2s-1)/8h黑暗),之后进行显微镜分析。如Lindsey&Gallois,1990(Journal ofexperimental botany,41(5),529-536)所述进行甜菜愈伤组织的稳定转化(通过卡那霉素选择)。在大约2个月后(24℃,16h光照(55μmolm-2s-1)/8h黑暗),经显微镜分析愈伤组织细胞。
CENH3转基因的克隆与世代
为了产生35S::RFP-CENH3融合构建体,使用如下引物从甜菜cDNA中扩增CENH3:BvCENH3-cds1:GGATCCATGAGAGTTAAACACACTGC(SEQ ID No.16),BvCENH3-cds2:GGATCCTGTTCAGTTACCATCCCCTC(SEQ ID No.17),克隆进含有35Spro、RFP和35S-终止子表达盒的载体中。对于含有在CENH3编码序列内导致F106和I106代替L106的突变的构建体,含有35S::RFP-CENH3表达盒的上述质粒用作进行基于引物的诱变的模板。接近希望的突变位置的PstI位点用于将CENH3分成两部分。通过引物中的突变,整合希望的突变:
BvCENH3_mut_Fw:ATGGATCCATGAGAGTTAAACACACTGC(SEQ ID No.18),
BvCENH3_L->F_Rv:CTCTGCAGCCTCTTGAAGGGCCATAAAAGC(SEQ ID No.19),
BvCENH3_L->I_Rv:CTCTGCAGCCTCTTGAAGGGCCATAATAGC(SEQ ID No.20)。
所得表达盒(35Spro,RFP-(突变的)CENH3和35S-终止子)通过测序核实。
在甜菜中分析CENH3结合
为了分析CENH3与突变的CENH3的结合,使用Axio Imager M2显微镜***的C-Apo63x/1.2W Korr物镜和软件ZEN(Zeiss,Germany)分析叶或愈伤组织材料。
实施例4:其它CENH3突变体的鉴别和检测
为了鉴别CENH3的内源基因内其它单一点突变,其导致翻译的CENH3序列的氨基酸取代或者缺失一或多个氨基酸。即使Ravi和Chan2010仅强调N末端结构域的特殊重要性,但是在另一部分CENH3如α2-螺旋中对于突变的上述研究(实施例1-3)表明CENH3的CATD结构域的修饰可导致CENH3与DNA的结合能力不稳定。因此,关注点是CATD内、特别是环1和α2-螺旋中其它突变的鉴别。此外,应证实由于物种之间CATD结构域的高水平保守所致,鉴别的突变具有赋予不同植物物种单倍体诱导物生物活性的潜力。
对于两种其它单子叶植物即玉米和高粱及对于两种双子叶植物即甘蓝型油菜和甜菜产生了具有高突变比率的TILLING种群。为了筛选内源CENH3基因中导致翻译的CENH3蛋白质的CATD结构域中至少一个氨基酸取代或者缺失至少一个氨基酸的突变,如上文针对大麦示例描述(实施例1)分别产生覆盖CENH3基因的全部外显子以及部分相应内含子的扩增子,通过Sanger测序方法分析每个植物物种的1000-10000个体。此外,使用特异性PCR检测M2甜菜植物的突变。
进一步地,CENH3基因内鉴别的突变对于编码的蛋白质的初级结构和二级结构的影响使用Prof软件等评估(Rost,B.and Sander,C.(1994a).Combining evolutionaryinformation and neural networks to predict protein secondarystructure.Proteins,19(1),55-72.Rost,B.and Sander,C.(1994b).Conservation andprediction of solvent accessibility in protein families.Proteins,20(3),216-26.Rost,B.,Casadio,R.,Fariselli,P.,and Sander,C.(1995).Transmembrane helicespredicted at 95%accuracy.Protein Sci,4(3),521-33.)。
纯合突变体的突变的CENH3的无功能性(non-functionality)例如如上述通过用CENH3特异性抗体对着丝粒进行免疫染色进行检测(实施例1和2)。鉴别的TILLING株系示出突变的CENH3的显著降低或受损的着丝粒荷载。具有对于这种突变杂合的基因组的植物是可存活的,且未观测到植物***、周期值和生长与相应野生型植物相当。
通过将突变体植物与相同物种的检测植物杂交评估鉴别的突变体中单倍体诱导物的生物活性。检测植物携带野生型形式CENH3。检测单倍体诱导的母本以及父本性能。为此,突变体植物在与检测植物杂交中用作胚珠亲本或者用作花粉亲本。如果使用的检测植物株系携带隐性非-CENH3突变,则从这个杂交中推定的单倍体后代可以迅速确定。如此,单倍体植物示出隐性表型。例如,在玉米中可以使用突变glossy的表现(Mutants of maize,Neuffer,MG et al.1997.Cold Spring Harbor Laboratory,New York)。如果使用的检测植物株系携带隐性非-CENH3突变,这些杂交的单倍体后代可以迅速确定。如此,单倍体植物示出隐性表型。例如在玉米中可以使用突变glossy的表现(Mutants of maize,Neuffer,MGet al.1997.Cold Spring Harbor Laboratory,New York)。
此外,使用诱导物的有丝***和减数***的细胞发生学分析表明作为单倍体诱导物的突变体的适合性,及纯合性通过使用分子标记、检测植物的多形性及潜在的诱导物确定。这种单倍体可以经细胞遗传学检测。
下列表格示出赋予所研究的植物物种单倍体诱导物生物活性的错义和缺失突变。
表4:来源于甘蓝型油菜的CENH3的突变(aa:氨基酸,nd:未确定,y:是,n:否)。氨基酸取代以X#Y示出,即在位置#氨基酸X(单字母代码)由氨基酸Y取代。
表5:来源于玉米的CENH3的突变(aa:氨基酸,nd:未确定,y:是,n:否)。氨基酸取代以X#Y示出,即在位置#氨基酸X(单字母代码)由氨基酸Y取代。
表6:来源于高粱的CENH3的突变(aa:氨基酸,nd:未确定,y:是,n:否)。氨基酸取代以X#Y示出,即在位置#氨基酸X(单字母代码)由氨基酸Y取代。
表7:来源于甜菜的CENH3的突变(aa:氨基酸,nd:未确定,y:是,n:否)。氨基酸取代以X#Y示出,即在位置#氨基酸X(单字母代码)由氨基酸Y取代。
与检测植物杂交,具有突变的内源CENH3的TILLING植物产生至少0.5%的单倍体后代。例如,在甘蓝型油菜中,突变C153Y和A154V示出在0.5%-1%之间的诱导率。在少数情况中,可以达到2%或更高的诱导率。通常地,如果检测植物在杂交中用作雄性亲本则诱导率较高。
此外,杂交结果证实鉴别的突变在其它植物物种也可以具有功能性。因此,在SEQID No.1所示共有序列的氨基酸位置4的突变,由此亮氨酸被取代为苯丙氨酸,如实施1-3所示在大麦(L92F)中产生诱导活性,在甘蓝型油菜(L132F)中也如此。因此,通过如TILLING、诱变或者基因组编辑(例如CRISPR/Cas,TALENs,锌指核酸酶等)等技术可以将突变导入其它植物物种中。此外,单倍体诱导物的生物活性和效力通过在一个植物中组合不同的鉴别的突变和/或修饰单倍体诱导物的遗传背景而可以进一步改良。不同突变的组合可以通过基因组编辑有效实现,或者突变体单倍体诱导物再次被诱变。
序列表
<110> KWS种子欧洲股份公司
<120> 单倍体植物的产生
<130> KWS0219PCT
<150> EP 14182719.6
<151> 2014-08-28
<150> EP 14004389.4
<151> 2014-12-23
<160> 97
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid consensus sequence of the Alpha-2-helix of the CENH3
<400> 1
Ala Glu Ala Leu Leu Ala Leu Gln Glu Ala Ala Glu Asp Phe Leu Val
1 5 10 15
His Leu Phe Glu Asp Ala Met Leu Cys Ala Ile His Ala
20 25
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 2
aggcagggtc tcaattcctt 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
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<223> Primer
<400> 3
gtcccatcat ccatcgtctt 20
<210> 4
<211> 20
<212> DNA
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<400> 4
cccacttcct tgttgtggac 20
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<212> DNA
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ggcgataaat gtatcttgca ttc 23
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<211> 20
<212> DNA
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tggtagcaac cagagctacg 20
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actggcatgt ttccttctgc 20
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cggacggagg gagtatttct 20
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ggacatgccc aaagaaagtg 20
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<212> DNA
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gccagcgagt actcctacaa g 21
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<212> DNA
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ttgagttacc agccaccact c 21
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<212> DNA
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gtcatgcact gtgtcttgca 20
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<212> DNA
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tgctaagatc ggataactgt gg 22
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<212> DNA
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<400> 14
tgctcctgaa caaactgaac c 21
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<211> 19
<212> DNA
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<400> 15
gtggccgtca gtacaatcg 19
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<211> 26
<212> DNA
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<400> 16
ggatccatga gagttaaaca cactgc 26
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<212> DNA
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ggatcctgtt cagttaccat cccctc 26
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<212> DNA
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atggatccat gagagttaaa cacactgc 28
<210> 19
<211> 30
<212> DNA
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<400> 19
ctctgcagcc tcttgaaggg ccataaaagc 30
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ctctgcagcc tcttgaaggg ccataatagc 30
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caatgatagc tgcaccacca actg 24
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<212> DNA
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ctagctgccc ttccacctct cca 23
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<212> DNA
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<223> Primer
<400> 23
agtcggtcaa ttttctcatc cc 22
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<212> DNA
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<400> 24
ctctgtagcc tcttgaactg c 21
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<212> DNA
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<400> 25
gccattgtcg aacaagaagg 20
<210> 26
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<212> DNA
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taacacggtg cgaatgaatg 20
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gacagctgaa gcatttgttg ctcttc 26
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gacagctgaa gctattgttg ctcttc 26
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caacgattga tttggggagg g 21
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ggtgcgattt ctccagcagt aaaaatc 27
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ctgagaagat gaagcaccgg cgatat 26
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<400> 32
aacttttgcc atcctcgttt ctgtt 25
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<211> 420
<212> DNA
<213> Hordeum vulgare
<400> 33
atggctcgca cgaagaaaac ggtggcggcg aaggagaagc gccccccttg ctccaagtcg 60
gagccgcagt cgcagccgaa gaagaaggag aagcgggcgt accggttccg gccgggcacg 120
gtggcgctgc gggagatccg gaagtaccgc aagtccacca atatgctcat cccctttgcg 180
cccttcgtcc gcctggtcag ggacatcgcc gacaacttga cgccattgtc gaacaagaag 240
gagagcaagc cgacgccatg gactcctctc gcgctcctct cgttgcaaga gtctgcagag 300
tatcacttgg tcgatctatt tggaaaggca aatctgtgtg ccattcattc gcaccgtgtt 360
accatcatgc taaaggacat gcagcttgcg aggcgtatcg ggacgagaag cctttggtga 420
<210> 34
<211> 139
<212> PRT
<213> Hordeum vulgare
<400> 34
Met Ala Arg Thr Lys Lys Thr Val Ala Ala Lys Glu Lys Arg Pro Pro
1 5 10 15
Cys Ser Lys Ser Glu Pro Gln Ser Gln Pro Lys Lys Lys Glu Lys Arg
20 25 30
Ala Tyr Arg Phe Arg Pro Gly Thr Val Ala Leu Arg Glu Ile Arg Lys
35 40 45
Tyr Arg Lys Ser Thr Asn Met Leu Ile Pro Phe Ala Pro Phe Val Arg
50 55 60
Leu Val Arg Asp Ile Ala Asp Asn Leu Thr Pro Leu Ser Asn Lys Lys
65 70 75 80
Glu Ser Lys Pro Thr Pro Trp Thr Pro Leu Ala Leu Leu Ser Leu Gln
85 90 95
Glu Ser Ala Glu Tyr His Leu Val Asp Leu Phe Gly Lys Ala Asn Leu
100 105 110
Cys Ala Ile His Ser His Arg Val Thr Ile Met Leu Lys Asp Met Gln
115 120 125
Leu Ala Arg Arg Ile Gly Thr Arg Ser Leu Trp
130 135
<210> 35
<211> 420
<212> DNA
<213> Hordeum vulgare (Tilling Line 4528 Mutant)
<400> 35
atggctcgca cgaagaaaac ggtggcggcg aaggagaagc gccccccttg ctccaagtcg 60
gagccgcagt cgcagccgaa gaagaaggag aagcgggcgt accggttccg gccgggcacg 120
gtggcgctgc gggagatccg gaagtaccgc aagtccacca atatgctcat cccctttgcg 180
cccttcgtcc gcctggtcag ggacatcgcc gacaacttga cgccattgtc gaacaagaag 240
gagagcaagc cgacgccatg gactcctctc gcgttcctct cgttgcaaga gtctgcagag 300
tatcacttgg tcgatctatt tggaaaggca aatctgtgtg ccattcattc gcaccgtgtt 360
accatcatgc taaaggacat gcagcttgcg aggcgtatcg ggacgagaag cctttggtga 420
<210> 36
<211> 139
<212> PRT
<213> Hordeum vulgare (Tilling Line 4528 Mutant)
<400> 36
Met Ala Arg Thr Lys Lys Thr Val Ala Ala Lys Glu Lys Arg Pro Pro
1 5 10 15
Cys Ser Lys Ser Glu Pro Gln Ser Gln Pro Lys Lys Lys Glu Lys Arg
20 25 30
Ala Tyr Arg Phe Arg Pro Gly Thr Val Ala Leu Arg Glu Ile Arg Lys
35 40 45
Tyr Arg Lys Ser Thr Asn Met Leu Ile Pro Phe Ala Pro Phe Val Arg
50 55 60
Leu Val Arg Asp Ile Ala Asp Asn Leu Thr Pro Leu Ser Asn Lys Lys
65 70 75 80
Glu Ser Lys Pro Thr Pro Trp Thr Pro Leu Ala Phe Leu Ser Leu Gln
85 90 95
Glu Ser Ala Glu Tyr His Leu Val Asp Leu Phe Gly Lys Ala Asn Leu
100 105 110
Cys Ala Ile His Ser His Arg Val Thr Ile Met Leu Lys Asp Met Gln
115 120 125
Leu Ala Arg Arg Ile Gly Thr Arg Ser Leu Trp
130 135
<210> 37
<211> 537
<212> DNA
<213> Arabidopsis thaliana
<400> 37
atggcgagaa ccaagcatcg cgttaccagg tcacaacctc ggaatcaaac tgatgccgcc 60
ggtgcttcat cttctcaggc ggcaggtcca actacgaccc cgacaaggag aggcggtgaa 120
ggtggagata atactcaaca aacaaatcct acaacttcac cagctactgg tacaaggaga 180
ggggctaaga gatccagaca ggctatgcca cgaggctcac agaagaagtc ttatcgatac 240
aggccaggaa ccgttgctct aaaagagatt cgccatttcc agaagcagac aaaccttctt 300
attccggctg ccagtttcat aagagaagtg agaagtataa cccatatgtt ggcccctccc 360
caaatcaatc gttggacagc tgaagctctt gttgctcttc aagaggcggc agaagattac 420
ttggttggtt tgttctcaga ttcaatgctc tgtgctatcc atgcaagacg tgttactcta 480
atgagaaaag actttgaact tgcacgccgg cttggaggaa aaggcagacc atggtga 537
<210> 38
<211> 178
<212> PRT
<213> Arabidopsis thaliana
<400> 38
Met Ala Arg Thr Lys His Arg Val Thr Arg Ser Gln Pro Arg Asn Gln
1 5 10 15
Thr Asp Ala Ala Gly Ala Ser Ser Ser Gln Ala Ala Gly Pro Thr Thr
20 25 30
Thr Pro Thr Arg Arg Gly Gly Glu Gly Gly Asp Asn Thr Gln Gln Thr
35 40 45
Asn Pro Thr Thr Ser Pro Ala Thr Gly Thr Arg Arg Gly Ala Lys Arg
50 55 60
Ser Arg Gln Ala Met Pro Arg Gly Ser Gln Lys Lys Ser Tyr Arg Tyr
65 70 75 80
Arg Pro Gly Thr Val Ala Leu Lys Glu Ile Arg His Phe Gln Lys Gln
85 90 95
Thr Asn Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val Arg Ser
100 105 110
Ile Thr His Met Leu Ala Pro Pro Gln Ile Asn Arg Trp Thr Ala Glu
115 120 125
Ala Leu Val Ala Leu Gln Glu Ala Ala Glu Asp Tyr Leu Val Gly Leu
130 135 140
Phe Ser Asp Ser Met Leu Cys Ala Ile His Ala Arg Arg Val Thr Leu
145 150 155 160
Met Arg Lys Asp Phe Glu Leu Ala Arg Arg Leu Gly Gly Lys Gly Arg
165 170 175
Pro Trp
<210> 39
<211> 537
<212> DNA
<213> Arabidopsis thaliana
<400> 39
atggcgagaa ccaagcatcg cgttaccagg tcacaacctc ggaatcaaac tgatgccgcc 60
ggtgcttcat cttctcaggc ggcaggtcca actacgaccc cgacaaggag aggcggtgaa 120
ggtggagata atactcaaca aacaaatcct acaacttcac cagctactgg tacaaggaga 180
ggggctaaga gatccagaca ggctatgcca cgaggctcac agaagaagtc ttatcgatac 240
aggccaggaa ccgttgctct aaaagagatt cgccatttcc agaagcagac aaaccttctt 300
attccggctg ccagtttcat aagagaagtg agaagtataa cccatatgtt ggcccctccc 360
caaatcaatc gttggacagc tgaagctatt gttgctcttc aagaggcggc agaagattac 420
ttggttggtt tgttctcaga ttcaatgctc tgtgctatcc atgcaagacg tgttactcta 480
atgagaaaag actttgaact tgcacgccgg cttggaggaa aaggcagacc atggtga 537
<210> 40
<211> 178
<212> PRT
<213> Arabidopsis thaliana
<400> 40
Met Ala Arg Thr Lys His Arg Val Thr Arg Ser Gln Pro Arg Asn Gln
1 5 10 15
Thr Asp Ala Ala Gly Ala Ser Ser Ser Gln Ala Ala Gly Pro Thr Thr
20 25 30
Thr Pro Thr Arg Arg Gly Gly Glu Gly Gly Asp Asn Thr Gln Gln Thr
35 40 45
Asn Pro Thr Thr Ser Pro Ala Thr Gly Thr Arg Arg Gly Ala Lys Arg
50 55 60
Ser Arg Gln Ala Met Pro Arg Gly Ser Gln Lys Lys Ser Tyr Arg Tyr
65 70 75 80
Arg Pro Gly Thr Val Ala Leu Lys Glu Ile Arg His Phe Gln Lys Gln
85 90 95
Thr Asn Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val Arg Ser
100 105 110
Ile Thr His Met Leu Ala Pro Pro Gln Ile Asn Arg Trp Thr Ala Glu
115 120 125
Ala Ile Val Ala Leu Gln Glu Ala Ala Glu Asp Tyr Leu Val Gly Leu
130 135 140
Phe Ser Asp Ser Met Leu Cys Ala Ile His Ala Arg Arg Val Thr Leu
145 150 155 160
Met Arg Lys Asp Phe Glu Leu Ala Arg Arg Leu Gly Gly Lys Gly Arg
165 170 175
Pro Trp
<210> 41
<211> 537
<212> DNA
<213> Arabidopsis thaliana
<400> 41
atggcgagaa ccaagcatcg cgttaccagg tcacaacctc ggaatcaaac tgatgccgcc 60
ggtgcttcat cttctcaggc ggcaggtcca actacgaccc cgacaaggag aggcggtgaa 120
ggtggagata atactcaaca aacaaatcct acaacttcac cagctactgg tacaaggaga 180
ggggctaaga gatccagaca ggctatgcca cgaggctcac agaagaagtc ttatcgatac 240
aggccaggaa ccgttgctct aaaagagatt cgccatttcc agaagcagac aaaccttctt 300
attccggctg ccagtttcat aagagaagtg agaagtataa cccatatgtt ggcccctccc 360
caaatcaatc gttggacagc tgaagcattt gttgctcttc aagaggcggc agaagattac 420
ttggttggtt tgttctcaga ttcaatgctc tgtgctatcc atgcaagacg tgttactcta 480
atgagaaaag actttgaact tgcacgccgg cttggaggaa aaggcagacc atggtga 537
<210> 42
<211> 178
<212> PRT
<213> Arabidopsis thaliana
<400> 42
Met Ala Arg Thr Lys His Arg Val Thr Arg Ser Gln Pro Arg Asn Gln
1 5 10 15
Thr Asp Ala Ala Gly Ala Ser Ser Ser Gln Ala Ala Gly Pro Thr Thr
20 25 30
Thr Pro Thr Arg Arg Gly Gly Glu Gly Gly Asp Asn Thr Gln Gln Thr
35 40 45
Asn Pro Thr Thr Ser Pro Ala Thr Gly Thr Arg Arg Gly Ala Lys Arg
50 55 60
Ser Arg Gln Ala Met Pro Arg Gly Ser Gln Lys Lys Ser Tyr Arg Tyr
65 70 75 80
Arg Pro Gly Thr Val Ala Leu Lys Glu Ile Arg His Phe Gln Lys Gln
85 90 95
Thr Asn Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val Arg Ser
100 105 110
Ile Thr His Met Leu Ala Pro Pro Gln Ile Asn Arg Trp Thr Ala Glu
115 120 125
Ala Phe Val Ala Leu Gln Glu Ala Ala Glu Asp Tyr Leu Val Gly Leu
130 135 140
Phe Ser Asp Ser Met Leu Cys Ala Ile His Ala Arg Arg Val Thr Leu
145 150 155 160
Met Arg Lys Asp Phe Glu Leu Ala Arg Arg Leu Gly Gly Lys Gly Arg
165 170 175
Pro Trp
<210> 43
<211> 465
<212> DNA
<213> Beta vulgaris
<400> 43
atgagagtta aacacactgc tgccaggaaa tcaaccacca atggtcctcg ttcaaaggct 60
cagaaatctc cgcgcagttt gcaatcacca caatcgcctt ctagtagttc aaagcgcaaa 120
tcacgcagaa acactgatgc aactcctcaa aagaagaagg cttaccgccg taagccgggc 180
actgtggcac tctgggaaat acgcaaattt cagaagtcat tcaagccctt gattcctgct 240
gcgcctttca ttcgaacagt gagagagatt actcaccagt ttgctcctta tgttggtcgt 300
tggcaagctg aagctctgat ggcccttcaa gaggctgcag agaattttat tgtccgtttg 360
tttgaagatg gtatgctttg tgcaattcat gccaaacgag ttacactcat gaaaaaggat 420
ttggagctcg cgcgaaggat tgggggcaga gagaggggat ggtaa 465
<210> 44
<211> 154
<212> PRT
<213> Beta vulgaris
<400> 44
Met Arg Val Lys His Thr Ala Ala Arg Lys Ser Thr Thr Asn Gly Pro
1 5 10 15
Arg Ser Lys Ala Gln Lys Ser Pro Arg Ser Leu Gln Ser Pro Gln Ser
20 25 30
Pro Ser Ser Ser Ser Lys Arg Lys Ser Arg Arg Asn Thr Asp Ala Thr
35 40 45
Pro Gln Lys Lys Lys Ala Tyr Arg Arg Lys Pro Gly Thr Val Ala Leu
50 55 60
Trp Glu Ile Arg Lys Phe Gln Lys Ser Phe Lys Pro Leu Ile Pro Ala
65 70 75 80
Ala Pro Phe Ile Arg Thr Val Arg Glu Ile Thr His Gln Phe Ala Pro
85 90 95
Tyr Val Gly Arg Trp Gln Ala Glu Ala Leu Met Ala Leu Gln Glu Ala
100 105 110
Ala Glu Asn Phe Ile Val Arg Leu Phe Glu Asp Gly Met Leu Cys Ala
115 120 125
Ile His Ala Lys Arg Val Thr Leu Met Lys Lys Asp Leu Glu Leu Ala
130 135 140
Arg Arg Ile Gly Gly Arg Glu Arg Gly Trp
145 150
<210> 45
<211> 465
<212> DNA
<213> Beta vulgaris
<400> 45
atgagagtta aacacactgc tgccaggaaa tcaaccacca atggtcctcg ttcaaaggct 60
cagaaatctc cgcgcagttt gcaatcacca caatcgcctt ctagtagttc aaagcgcaaa 120
tcacgcagaa acactgatgc aactcctcaa aagaagaagg cttaccgccg taagccgggc 180
actgtggcac tctgggaaat acgcaaattt cagaagtcat tcaagccctt gattcctgct 240
gcgcctttca ttcgaacagt gagagagatt actcaccagt ttgctcctta tgttggtcgt 300
tggcaagctg aagcttttat ggcccttcaa gaggctgcag agaattttat tgtccgtttg 360
tttgaagatg gtatgctttg tgcaattcat gccaaacgag ttacactcat gaaaaaggat 420
ttggagctcg cgcgaaggat tgggggcaga gagaggggat ggtaa 465
<210> 46
<211> 154
<212> PRT
<213> Beta vulgaris
<400> 46
Met Arg Val Lys His Thr Ala Ala Arg Lys Ser Thr Thr Asn Gly Pro
1 5 10 15
Arg Ser Lys Ala Gln Lys Ser Pro Arg Ser Leu Gln Ser Pro Gln Ser
20 25 30
Pro Ser Ser Ser Ser Lys Arg Lys Ser Arg Arg Asn Thr Asp Ala Thr
35 40 45
Pro Gln Lys Lys Lys Ala Tyr Arg Arg Lys Pro Gly Thr Val Ala Leu
50 55 60
Trp Glu Ile Arg Lys Phe Gln Lys Ser Phe Lys Pro Leu Ile Pro Ala
65 70 75 80
Ala Pro Phe Ile Arg Thr Val Arg Glu Ile Thr His Gln Phe Ala Pro
85 90 95
Tyr Val Gly Arg Trp Gln Ala Glu Ala Phe Met Ala Leu Gln Glu Ala
100 105 110
Ala Glu Asn Phe Ile Val Arg Leu Phe Glu Asp Gly Met Leu Cys Ala
115 120 125
Ile His Ala Lys Arg Val Thr Leu Met Lys Lys Asp Leu Glu Leu Ala
130 135 140
Arg Arg Ile Gly Gly Arg Glu Arg Gly Trp
145 150
<210> 47
<211> 465
<212> DNA
<213> Beta vulgaris
<400> 47
atgagagtta aacacactgc tgccaggaaa tcaaccacca atggtcctcg ttcaaaggct 60
cagaaatctc cgcgcagttt gcaatcacca caatcgcctt ctagtagttc aaagcgcaaa 120
tcacgcagaa acactgatgc aactcctcaa aagaagaagg cttaccgccg taagccgggc 180
actgtggcac tctgggaaat acgcaaattt cagaagtcat tcaagccctt gattcctgct 240
gcgcctttca ttcgaacagt gagagagatt actcaccagt ttgctcctta tgttggtcgt 300
tggcaagctg aagctattat ggcccttcaa gaggctgcag agaattttat tgtccgtttg 360
tttgaagatg gtatgctttg tgcaattcat gccaaacgag ttacactcat gaaaaaggat 420
ttggagctcg cgcgaaggat tgggggcaga gagaggggat ggtaa 465
<210> 48
<211> 154
<212> PRT
<213> Beta vulgaris
<400> 48
Met Arg Val Lys His Thr Ala Ala Arg Lys Ser Thr Thr Asn Gly Pro
1 5 10 15
Arg Ser Lys Ala Gln Lys Ser Pro Arg Ser Leu Gln Ser Pro Gln Ser
20 25 30
Pro Ser Ser Ser Ser Lys Arg Lys Ser Arg Arg Asn Thr Asp Ala Thr
35 40 45
Pro Gln Lys Lys Lys Ala Tyr Arg Arg Lys Pro Gly Thr Val Ala Leu
50 55 60
Trp Glu Ile Arg Lys Phe Gln Lys Ser Phe Lys Pro Leu Ile Pro Ala
65 70 75 80
Ala Pro Phe Ile Arg Thr Val Arg Glu Ile Thr His Gln Phe Ala Pro
85 90 95
Tyr Val Gly Arg Trp Gln Ala Glu Ala Ile Met Ala Leu Gln Glu Ala
100 105 110
Ala Glu Asn Phe Ile Val Arg Leu Phe Glu Asp Gly Met Leu Cys Ala
115 120 125
Ile His Ala Lys Arg Val Thr Leu Met Lys Lys Asp Leu Glu Leu Ala
130 135 140
Arg Arg Ile Gly Gly Arg Glu Arg Gly Trp
145 150
<210> 49
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid consensus sequence of the loop1 of the CENH3
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa can be any naturally occurring amino acid
<400> 49
Thr Asn Phe Leu Ala Pro Xaa Glu Val Thr Arg Trp Thr
1 5 10
<210> 50
<211> 4001
<212> DNA
<213> Brassica napus
<220>
<221> Intron
<222> (1892)..(1972)
<220>
<221> Intron
<222> (2020)..(2104)
<220>
<221> Intron
<222> (2148)..(2208)
<220>
<221> Intron
<222> (2264)..(2358)
<220>
<221> Intron
<222> (2399)..(2507)
<220>
<221> Intron
<222> (2624)..(2755)
<220>
<221> Intron
<222> (2832)..(2933)
<220>
<221> Intron
<222> (3012)..(3091)
<400> 50
tgtccgggag gatccaccgg cggtggtcgt tactccattt taacttgatg tcttgaaagc 60
agaggacatg gtatggtggc ggcagaatct atttttagtt gttaattttt cttttcctct 120
gattcttttt atttttttcg aatgaactaa ctttgggttt attcagaaga attatcatct 180
aaaaactgat tcaataaaca aaataattta catatttcac aatgagccat tagtaaacaa 240
gtcgaaagtg aaaccaaatg ggaagagaac aattttaata aaaatatgtt ctaatttcct 300
actttttatg aattgaactc ccgaagagaa tggccgaaga acggagtaaa agctcaatga 360
ttgtaaaagc tatgtattct cttgctttga ggaaaaagct ttttgtttgc actcataggc 420
ctgatatgtt gtgatggctc tttacatatt gggtcttttg tggtctatta aacggttact 480
gaagaattag tttatcgcat ttaaaaaaaa atatttgaaa accacatatc taaaatctca 540
atatattatt tattgataga tgaaaagaaa aatagtttat aaaataatat taattggtaa 600
atggaagtca aaattttaca tttaatacat gactagttcg attctccggc tgaaacccat 660
ttaccactga gccaagacca cttgattata tatatcaagc tcttgttttt tgcattaaca 720
aaagttgcat ccaaaatttc aaaacaacaa ctaaattaat gtctttctat tttcaaagtt 780
ttatctccaa acctattaat agtttaattt ttttttttaa aattaggaaa ccggttaaaa 840
tatattttaa atacaaaaaa cttaaacaat gaatcattta tttatttatt cttcaaaatt 900
taaatatccg aacccggccc aaaatatccg aacccgaaca taaaatatcc gaacccgact 960
cgaagtgtag aaaatatccg aacgggtttt atacctttat actgaaatac cctatacgaa 1020
cccgaatgtg tatccgaacg ccccctacaa tatatgatca tcatttgtat cttgattgaa 1080
caaaaaaaaa gttaaactat tgatcacaaa attttcaatg tgagactttt accattttta 1140
gtcatttata gtcgttttta aaaattcaaa atataactta taagaaaaaa tctaattttt 1200
tttattatat gcttaatgtg attgtttaat ttcttttaat aatataaaat taaacaaaaa 1260
atgagaggtt aaaaaaattg ttatcaaata tgtattattc ataatcatta attgtcatat 1320
atatgttaat tatattaggt aatttcgtag tttttattta agaaaagaaa aaaatattat 1380
tttgtacact actaattaat ttgatagtta gtttaataaa aaatatatta tattattata 1440
tggaccaact tatttttcta aaaaaaaacc actgttttaa aaaccaaacc aactataaac 1500
cggagatata ccggattgag tggctaaaac actctttgta tatatgtgct gagcaaaccc 1560
tctgagtgag atggcgtgtt aagaagtagg aggaccattc atgcctctta tgagttgtag 1620
tctgtgtgta caaaaaagaa gcgttggtgt gaaagaaagc agaaggattt gaaaatcaaa 1680
aaaattgaag gagaagcggg aaaacaaata atctctccct ccgctttttt ttctccaaat 1740
aatcaatctc tcatttcatt tgttaaccca agtttttgat aattatttca aaggggttta 1800
tttatctttt attcctccgg cggcagtaag tagtaatcaa tggcgagaac caaacatttc 1860
gcttccaggg cacgagatcg caatcgaact agttagtact ctctctctct ctgccttttt 1920
tttgatattt attttctagg ttaaacccta atttggcatc tgaaatttgt agatgcgact 1980
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcgg tacgtcatct attttctttt 2040
cccgttttag gtttttacgc aaatctcgtt actgtttttt tgacgaatcg attgaaatgt 2100
gtagaccccg acgagaagag aaggcagcca aggagaagct caacagagtg agtctttcta 2160
tttcattttc tgagatccat gaatcctttt catctctcgt gtgttgtgac atgaatcaat 2220
tgcagcagca actcctacta cgactccacc agccggtaga aaagtaagtt acatttccat 2280
ttcacaccat tcatttgctt ctttatcaac aaactgctct ctcatctgtt ttttttgttt 2340
tgttttggtt ttgtgaagaa aggagggact aagcgaacta aacaagctat gcctaaaagt 2400
tagtgacaga ttttaaaatc tctattttgg atcatcattc tctcaggaca tgtctatttg 2460
catttgttct tattatgtct gtctgtctgt ctttgtcccc cttgtaggtt ccaacaagaa 2520
gaagacattc cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa 2580
gaccaccaaa cttcttatcc ctgccgctag tttcatccga gaagttagta atgaactttg 2640
ttattcatac attcccgctt acttgttttc aatgactctg caattactga tatagaattt 2700
ggagcaacca ttatggggtg atttctctaa ctacaaatta ctaatactat cccaggtgag 2760
aagtgtcacc cagatctttg cccctcccga tgttacccgt tggactgctg aagctcttat 2820
ggctattcaa gaggtacgtg tactccttcc ctcttttgtt tcctattttc cacttgatgt 2880
ctaatttaaa ctgatcgttt tttttttata tttcttttgg tgtggggcgg ggcaggcggc 2940
tgaagatttt ttaattggct tgttctctga tgctatgctt tgcgctatcc atgcaaggcg 3000
tgttactcta agtaagtagt actccccaaa ataaggaaac ccattttata tacaacattg 3060
cctcatccat gtctgcttct cttcatatca gtgagaaaag attttgagct tgcacgccgt 3120
cttggaggaa aaggcagacc attgtgatcg tttcgcaggt tgtataactt tgttcactcc 3180
ttatgtcttg tcatttgtga tctgactgac actttctttt gaaacataac tgcttgattc 3240
aatatctagg ctgtaaaact tatccctcct tgtttactat cttatatgct ttttccttgg 3300
aattgatagt ttccattgag atttcacttg cacgaaacat atctgctttc tcaatatctc 3360
tcagtcttag aaagggctat tgactaaaag aaaagaaaat ttagaggaag atttgtaaag 3420
acatgtgttt agagagggct taattaaaaa cacacgcttc tgctagcctt gctatttgat 3480
tcccaatttc aacttttttc gaggcatatt ataaagtttt taaatgtact tggcacttca 3540
acttttataa tttatataac gattttattc taatagagca tttgtgattt catagtgttg 3600
tcatgaaact caagtaattc acaccgtccg atgttgctat tgtctaataa aatgttgaaa 3660
aaattgtcaa aacagaacaa aaaacaacat agttgtctct atggtataaa actatcacta 3720
agttgtctct atagtataat atttttcgca atcccaaaac taatttttct ttaatcaaat 3780
taaacataaa ctaaaaccat ttttaaaaag tttaatggaa aaagataaaa aaataaggta 3840
atctcgtaat gttttaaaaa ggaaaaaaaa tgtaaaaaca atttaaaaaa aagaacacac 3900
gacacagatc aaaaatatca tgtaatctaa ttgcatttgg tttctaaaat cttccaaaac 3960
tattctttta aaattctcta aggtaaaact tgattccaat a 4001
<210> 51
<211> 540
<212> DNA
<213> Artificial Sequence
<220>
<223> Brassica napus - cDNA coding for CENH3
<400> 51
atggcgagaa ccaaacattt cgcttccagg gcacgagatc gcaatcgaac taatgcgact 60
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcga ccccgacgag aagagaaggc 120
agccaaggag aagctcaaca gacaactcct actacgactc caccagccgg tagaaaaaaa 180
ggagggacta agcgaactaa acaagctatg cctaaaagtt ccaacaagaa gaagacattc 240
cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa gaccaccaaa 300
cttcttatcc ctgccgctag tttcatccga gaagtgagaa gtgtcaccca gatctttgcc 360
cctcccgatg ttacccgttg gactgctgaa gctcttatgg ctattcaaga ggcggctgaa 420
gattttttaa ttggcttgtt ctctgatgct atgctttgcg ctatccatgc aaggcgtgtt 480
actctaatga gaaaagattt tgagcttgca cgccgtcttg gaggaaaagg cagaccattg 540
<210> 52
<211> 180
<212> PRT
<213> Brassica napus
<400> 52
Met Ala Arg Thr Lys His Phe Ala Ser Arg Ala Arg Asp Arg Asn Arg
1 5 10 15
Thr Asn Ala Thr Ala Ser Ser Ser Ala Ala Ala Ala Glu Gly Pro Ser
20 25 30
Ala Thr Pro Thr Arg Arg Glu Gly Ser Gln Gly Glu Ala Gln Gln Thr
35 40 45
Thr Pro Thr Thr Thr Pro Pro Ala Gly Arg Lys Lys Gly Gly Thr Lys
50 55 60
Arg Thr Lys Gln Ala Met Pro Lys Ser Ser Asn Lys Lys Lys Thr Phe
65 70 75 80
Arg Tyr Lys Pro Gly Thr Val Ala Leu Arg Glu Ile Arg His Phe Gln
85 90 95
Lys Thr Thr Lys Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val
100 105 110
Arg Ser Val Thr Gln Ile Phe Ala Pro Pro Asp Val Thr Arg Trp Thr
115 120 125
Ala Glu Ala Leu Met Ala Ile Gln Glu Ala Ala Glu Asp Phe Leu Ile
130 135 140
Gly Leu Phe Ser Asp Ala Met Leu Cys Ala Ile His Ala Arg Arg Val
145 150 155 160
Thr Leu Met Arg Lys Asp Phe Glu Leu Ala Arg Arg Leu Gly Gly Lys
165 170 175
Gly Arg Pro Leu
180
<210> 53
<211> 3088
<212> DNA
<213> Sorghum bicolor
<220>
<221> Intron
<222> (377)..(487)
<220>
<221> Intron
<222> (514)..(618)
<220>
<221> Intron
<222> (662)..(1094)
<220>
<221> Intron
<222> (1217)..(1312)
<220>
<221> Intron
<222> (1391)..(2087)
<220>
<221> Intron
<222> (2164)..(2556)
<400> 53
catctctcac tgccatccgg gtccactact cccaacgttc ggcacgccag gtatagccgt 60
taccccggta ggccccactg gtacacggac aaaggttagc ggtcaccgcg aatcgtgaat 120
acttgtgact acggggtgct aattataaaa acgccgcaca tcctttcgtt tcgccatttc 180
accccccttc ccttcccgta gagaggaaaa aaacccaccg tcgacccgcc cggccgcccg 240
agagttctga atcgaaaccg tcggccgcga ccgcgagagc agcgcggggc gcccaccgtg 300
atggctcgaa ccaagcacca ggccgtgagg aagctgccgc agaagcccaa gaagaagctc 360
cagttcgagc gcgcaggtaa gcccgcgtcc ccgcgctgaa cccccctccg cctcgcgagc 420
agacgctgcc gctgctctcc gtcgcccctg gtgctaagcg cgttcctttt ttttccttct 480
tttgcaggtg gggcgagtac gtcggcgacc ccggtgagtg cgtgcgtgcg tgcgggaatt 540
ggttttagcc ctccttttgc ggtttcgcct tttgttgggc tggtctcact tgcttgcaat 600
ctgtttgatg gaatgcagga gaggaggaat gctgggaccg ggggaggagc cgcggctcgc 660
ggtgaggatc tctttgtcgt tgctgggttt gggaatttcc ggcgcgaaat tatgtggatt 720
tctaggttta tctgccgtct ttcttcttgt cttctctttt ggctctgggg tgagaagtta 780
gggtggttgg gcggacatgg tgcgttattt cgccgtatcg tttggtttgg tgctttctca 840
tccttttaat tccaacatgc cttgtaaaaa ttgcacaaga tttgtttttt catgcatgtc 900
tcagtgttgc taatttgctt ttccggttcg gttggtagaa ttcaatttct tggcgcaata 960
tgcatcttct tttgttgcaa catgagggcg aatgtgccag ttccatatgg gcgtcgcggt 1020
tttgaagtta ctaccttgct tgctcttcgt attataggcg tcattcacaa tagtatgttt 1080
tcttggagat gcagttgcac gggggcgtgt ggagaagaag catcgctggc gggcagggac 1140
tgtagcgctg cgggagatca ggaagtacca gaagtccact gagccgctca tcccctttgc 1200
gcccttcgta cgtgtggtgg gtgcatcttg taccaattgt tgtccactcc atagaatggg 1260
tttgttctgc agtctgtctg atggaaagtt attcttctga gaaaaaatgc aggtcaaaga 1320
gttaactgca ttcataacag actggaggat agggcgctac acccctgaag ccctccttgc 1380
gctgcaagag gtcagttatg aaacatgtct tgtgtatcag ttaagatcat cttctataga 1440
cataattgtt atcatgaagt ctttttctgt taatcggtct ggtactactt aataatcagg 1500
atttcagatt gctgcctttc ctagtggtgt agtcaaaagg gaatttaagt gctgttaggt 1560
actgtttgtt ttggtgtttt gaaccctgcc gcgatcggtt gttgttattc catgtttgtt 1620
tctgtggcag cggacgttca cggtgagatg ggatacgggc gtgtgaaaca tagttacggt 1680
ccatcttcat ggcttatcca tttacgctgc tcgtccgctc acttgttatg tgcggcaacc 1740
aaacttttgt tactagtgta actggtagcg ttgcaaatct ttccatttgc gttaccactc 1800
cctatgggag ccaaacagca ccttagtgta gattccattt gtattacttg agctagcttc 1860
cttgctattg gtgcctcgat tgtactgtta tgatcgaagt gctgaaaact ttgtcgcctg 1920
catagcatga ttagagaact tgagtttaca tttattcaat accttaagac tgcatttcgt 1980
atagataaat tatttttcct aattgttctg gttaactgtt ttaggtttcc atatttttgt 2040
atgtgtatca tttaaattat tgtgttgttt ttcctccctg tctacaggca gcagaattcc 2100
acttgataga actgtttgaa gtggcgaatc tgtgtgccat ccatgccaag cgcgtaacag 2160
tcagtaagtt atcactgaat gaactccttt tcctctgtac tattacgcct aatggagatg 2220
tgtgatgcat ttttggttac acgattcttt agtgattctg cttcagttgg atatgataaa 2280
tctagatgtt atttaaagtg gcaaattgct tacgagtgga aatagtaatg ttcaaatagt 2340
gaaaagtgca attaaacttt taataggcca ttatatggtt tgattgtcaa caaatgcatc 2400
aagaaatagt aaatattata acagttatgg cttagagagt ggacaaaaaa tcggtaatgg 2460
tgagctttgt ataaacacta aaactggctg agaaatctga taactcaagg atctatagga 2520
aatgtattat cctaaatgtt ttccttcctg ctgcagtgca aaaggacata caacttgcaa 2580
ggcgtatcgg aggaaggcgt tggtcgtgat atccattctg attctgatta ccttgttcgg 2640
gtggaatttg tttagaggag ttagacatta gtcttgttga atgctgtgca tggttcctaa 2700
tctgtttcac agttagtggg ctcttctggg atgatctgtt aacacctgtg gagtatgtta 2760
tgtaggaaac acctgaactg aacaacccaa agttgttttg gttgctcttc aaccatttgt 2820
ttgcttcaga gatcgattct aaactgcatg ctaattagtc tatggttgaa caaaaattat 2880
caaatataaa tgaaagtgat atagtagcaa aatccaaaaa aaaaaggatc caaacaaggc 2940
ctaaaatcat ggttctttct ccttttgaac tgggtgcaag tatggacagg cacagaagaa 3000
aaccgcctag caaaccgttt gttttttttt cttcgttgta ccacacgaca ctgttcgttc 3060
ctagttgcgc ctttttgttg tagaagtc 3088
<210> 54
<211> 471
<212> DNA
<213> Artificial Sequence
<220>
<223> Sorghum bicolor - cDNA coding for CENH3
<400> 54
atggctcgaa ccaagcacca ggccgtgagg aagctgccgc agaagcccaa gaagaagctc 60
cagttcgagc gcgcaggtgg ggcgagtacg tcggcgaccc cggagaggag gaatgctggg 120
accgggggag gagccgcggc tcgcgttgca cgggggcgtg tggagaagaa gcatcgctgg 180
cgggcaggga ctgtagcgct gcgggagatc aggaagtacc agaagtccac tgagccgctc 240
atcccctttg cgcccttcgt acgtgtggtc aaagagttaa ctgcattcat aacagactgg 300
aggatagggc gctacacccc tgaagccctc cttgcgctgc aagaggcagc agaattccac 360
ttgatagaac tgtttgaagt ggcgaatctg tgtgccatcc atgccaagcg cgtaacagtc 420
atgcaaaagg acatacaact tgcaaggcgt atcggaggaa ggcgttggtc g 471
<210> 55
<211> 157
<212> PRT
<213> Sorghum bicolor
<400> 55
Met Ala Arg Thr Lys His Gln Ala Val Arg Lys Leu Pro Gln Lys Pro
1 5 10 15
Lys Lys Lys Leu Gln Phe Glu Arg Ala Gly Gly Ala Ser Thr Ser Ala
20 25 30
Thr Pro Glu Arg Arg Asn Ala Gly Thr Gly Gly Gly Ala Ala Ala Arg
35 40 45
Val Ala Arg Gly Arg Val Glu Lys Lys His Arg Trp Arg Ala Gly Thr
50 55 60
Val Ala Leu Arg Glu Ile Arg Lys Tyr Gln Lys Ser Thr Glu Pro Leu
65 70 75 80
Ile Pro Phe Ala Pro Phe Val Arg Val Val Lys Glu Leu Thr Ala Phe
85 90 95
Ile Thr Asp Trp Arg Ile Gly Arg Tyr Thr Pro Glu Ala Leu Leu Ala
100 105 110
Leu Gln Glu Ala Ala Glu Phe His Leu Ile Glu Leu Phe Glu Val Ala
115 120 125
Asn Leu Cys Ala Ile His Ala Lys Arg Val Thr Val Met Gln Lys Asp
130 135 140
Ile Gln Leu Ala Arg Arg Ile Gly Gly Arg Arg Trp Ser
145 150 155
<210> 56
<211> 5834
<212> DNA
<213> Zea mays
<220>
<221> Intron
<222> (1820 )..(1917)
<220>
<221> Intron
<222> (1944 )..(2035)
<220>
<221> Intron
<222> (2085 )..(2239)
<220>
<221> Intron
<222> (2356 )..(2446)
<220>
<221> Intron
<222> (2525 )..(4530)
<220>
<221> Intron
<222> (4607 )..(5346)
<400> 56
ttatgtagag gcaattgcag tagtgcctct gttttagagt gtaactacag atttgtccct 60
atttttttag agtttgcgtg tttgtccctg ttttttcaaa tcaaactatt gtatacccct 120
actccattag ttatacttaa caatgttaag tcttgataaa aagacaaggg ataattggat 180
tagtgaccct gttttagagt gtaattatag ctttgcccga tgttttagac ttcacatgtt 240
tttatgacaa ttcaaattgt ttccataaca tcttaaatta ttttgacaac atttagaatt 300
gttttgcaat aatttaaatt atttccaaaa taaaaatatt ttgacaatta ttttatcaac 360
aaattaaatt atttttttta caaaataatt tgtcaaggta cttttttaaa attttgaaaa 420
taatcaaatt attgtaaata taatttgaat tgttgcgaaa ataatttgga ttgtcataaa 480
aacacgtaag tctaaaaatt aggcgtaaaa ctacaattat actctaaaaa ggtggtaatg 540
gcgtagttgt tccttgtcta ttttatcaag acatagcacc gtgcagtaca actaatggag 600
tagtgacata caacaatttt gttttaaata ataggggtaa atacgcaaag tctaaaaaac 660
agaggtaaat ctacaattac atgttaaaat agaggcatcg atacaattgt accttttata 720
tagcagcatg cgccctgttg ggatacaatt gtacctttca catgtcttct agatggttcc 780
caaccctttg gccaagatcg tacagataat attgcgagga gcccaaatca acggtgtcca 840
tatgttatgt tgatgtggat ggtttaccta ggcgcaaaag tgcgctggtt tcgtccgtac 900
aaatatactt taagtatggt tttgattttt ttctattttt cattttttaa ataaaacgag 960
acaatcaaat ctgatataaa aatcaaatga attataaata gagacggaaa gagtatatat 1020
atttgttttg ctattattta aagtattaaa agatagtgga cgaatgaacg tcctctatgt 1080
ttaaaagaac gttttagagg acgttgtgtt gttgaaggaa atatgaaaaa aaaatcttct 1140
gcatatttag aagggaggag cgtttacaca ttactttcgg gacttcaacc caaatatgtc 1200
aaggtttgtg agtggctcag tgcggaaaaa aaatcctata tataccagat gtaaacacta 1260
tcttttacag cctatcacat tcacatttag aggttcacaa agatagatca aaatttataa 1320
aataatcatt taatattttt tttattttat ttatatggat aagcagctgg tgtatgtgag 1380
gagctgtaaa agatattttt tacatccgag atgtaaagat tttttttaac tcaatgctgg 1440
ttaccggctg ggaggacgat gataaagaaa gcatctctca ctgcattccg ggcccactac 1500
tcaaacgttc ggcacgccag gttggcaggt agccgttaca tcgataggca ctcggccact 1560
cgcacgcaga caccacacca gtgtgctcag tgctcactgc tcaccataat aacgctgcac 1620
ctcttttcat ttcaccatct cctgccccct taaaaaaaag actcaccgtc gacacgccct 1680
cccgtcccga gagttctgaa tcgaaaccgt cggccacgag agcagtgcga ggcgcccacc 1740
gcgatggctc gaaccaagca ccaggccgtg aggaagacgg cggagaagcc caagaagaag 1800
ctccagttcg agcgctcagg taacccgggt cccgcgctcc cccccgcttc gcaagcagac 1860
gctgtcgctt ctctccgacc ctggtgctaa gcacgttcct tgttccgtct tttgcaggtg 1920
gtgcgagtac ctcggcgacg ccggtgagcg cgtgcgtgcg gggatcagtt ccctcctttt 1980
gccttttttt gttgggctgc tcttacttgc ttgcaagctg tttgatggaa tgcaggaaag 2040
ggctgctggg accgggggaa gagcggcgtc tggaggtgac tcaggtgagg acctatttgt 2100
cgttgctgga tgctgggttt cgcttgcaat ctaattttgt tgcaagatga gggcgaatgt 2160
gccagttcca tgtgggtgtc atggtctcgg agttactacc ttaattgctc accatagtat 2220
gttttcttaa aaaaaacagt taagaagacg aaaccacgcc accgctggcg gccagggact 2280
gtagcgctgc gggagatcag gaagtaccag aagtccactg aaccgctcat cccctttgcg 2340
cctttcgtcc gtgtggtggg tgcaggcgtg tttgtcctct gcatagtatg gggttgttcc 2400
gcattctgtc taatggaaag ttattcttct gagaaaaaaa atgcaggtga gggagttaac 2460
caatttcgta acaaacggga aagtagagcg ctataccgca gaagccctcc ttgcgctgca 2520
agaggtcagt tatgaaaaat gtcttatctc tctgttaaga tcctcttcat atacatagtt 2580
gctattgcta tcgtgaagtc ttttttttct gttaattggt ctggtactac ttactagtca 2640
ggatttcata ttgcggtttt tcctagtggt gtgtagttaa aaagtagttt aattgctttt 2700
agttaaaagg ggtgttcagg gctaaagatc aactatgaga aaacagaaat tttcccaatt 2760
cgatacccga cagcattatg gcctgcgcta atggaggtgt ttccgggcaa atactctagc 2820
ctacctggga agtaccttgg gttgcccctt catttcagga aagtaaaaag gaatgatctt 2880
caacctctaa tcgaaaaaat caacaacagg ctggccttgc tggaaaggca agatgttgtc 2940
caaggctggt atagaaactc ttgtaaaatc gatgctatcc gcacaaccaa tctaccatct 3000
aatggttttt ccacctcaaa aatggctgct gcaaacaatt gacaaaatac gaagaaactt 3060
cctgtggaga gggagcaatc cagaagtttg cagcgggggt cactgcctcg tcaactggcc 3120
cgtaacttgc ctcccaaaga acaagggagg tcttggaatt ctggaccttg atcgttttgc 3180
gagggggcta agactaagat ggctgtggct acgatggaag agcaaagata gggcgtggac 3240
tgccttgaag cttccttgtg acaaaactga tgaagatctc ttcaatgctt ccacaactgt 3300
cacggtaggc aatggaaaga tagctgaatt ctggaattct agttggatcc aaggccaagc 3360
ccctaagaac attgcgccaa cactgttcaa gaaggaaaag aggaagaaca tcacggtcgc 3420
caaagcgctc actaacaaca attggattcg tttatgctca ccatacacgg gtgaggggga 3480
gtttagagag gtcgtctctc tttggcaggc cataggtaac atgcaagagc ttaacggttt 3540
ggaagacaac atctcttgga gatggacggc agatgggcag tacagtgcta gcagtgcata 3600
caaaatccag ttcgcatcca atttcactaa aatgaacctc tgccctattt ggaaggctaa 3660
agtggaaccg aaatgccgat tttttgcttg gacactactt cataagagaa ttctgactgc 3720
cgataacctt cataaaagag gttgcaactc agcctcagaa acaattcccc acttatgcaa 3780
ggattgcccc tttagtagag aggtgtggaa caaagttttg tctcgggcca actttccttt 3840
actgactggg tctcccagtg acacttcttt gtatgattgg tggacggaca tgtgcagcct 3900
ttgcagcaga caggcaagaa gaggtttcga cggtctgcta tttcactttt ggtggaactt 3960
atggctggaa agaaataaca gaatctttca aaggcagcgt agaagtgtag atcaagttgc 4020
tctggcagtc aaggattatg ctagtagctg aagtctagtt ggtttggact agtggttttg 4080
ttgcttttct ttttaatttc tttttagttc tttttatgtt gttttcgttt ccttaagttg 4140
cttggagtct gtattatcct ctttcttcta atatagatcg gagcgacaaa ccttttgccc 4200
cttcctttca aaaaaaagtt aaaagggaat ttaactgctt tcctagtggt gtagttaaaa 4260
tggatttcat attgcggcct ttcctagctt gcttgctatt gattggacta tagtgatcca 4320
aatgctgata actttgtcgc ttgtgtaggc atggttagag agcttagagt ttgcatttat 4380
tcaatacctt gagactgcat ttcatataca taaattattc atgattattt cttttctcta 4440
tttgttctgg ttaattaaga gttttaggtt tccatatttt tgtacgtgca tcatttaaat 4500
tcttgtattg tttttcgttc ttgtctacag gcagcagaat tccacttgat agaactgttt 4560
gaaatggcga atctgtgtgc catccatgcc aagcgtgtca caatcagtaa gttatcactg 4620
agtgaactcc tttttctctg tagcattact cctaatgaat atgtgtgatg cattttggtt 4680
gcacgattct ttagtgattc tgcttcagat ggatatgata aatctagatg ttattttgaa 4740
gtggcgaatt gcttacgagc ggaaatagta atgttcaaat agcgcaaagt gcaactgttg 4800
acttttagta ggccatttat atggtttgat taccaacaaa tacgtcaatc atatgatttg 4860
attatcaaca aaggaatcag ctatatggtt tgattatcaa caaaggaatc agctaggttt 4920
gcttatcaac attcaacaaa ggcatcaagt aatactccat ccgtttcaat ttataattcg 4980
tttgactttt tttatctaag tttgatcggc tcgacttatt aaaaaaaatc ataattattg 5040
ttaatttttg ttgtgatatt gtttagtata atatacttta aatgtgactt tgagtttttc 5100
attttttcgc aaaaaaaaat gaataggacg agccggtcaa acgtgacaca aaaaagtcaa 5160
acgaattata atttgggaca cacggagtag taaataatgt aacaacttag agagtgggac 5220
aaaaaaatct ctagtggtgc taaatttagt tcagctttgt ataaacacaa gcattgattg 5280
agaaatctga caactcaagg atctgtagga aatgtgttac cctaaatgtt ttccttactg 5340
atgcagtgca aaaggacata caacttgcaa ggcgtatcgg aggaaggcgt tgggcatgat 5400
atataatatc cattctgatt gcatcattct tgtgaatttg tttgtaggag ctagacatta 5460
gtgttgttga atgctgcatg gttcctaatc cttttcgcag tctaacatct gtggagttag 5520
tatgttacat ggcaacagct gaacatctgt ggactatatg gcaacagccg aagattgtgt 5580
ctgtgggata actggttgtt ttggttgctc ttcagtagtt tgtttgcttc aggtaaccat 5640
gctgcgaact atgatgtttt cattctcggt ttgcttcagc taaccgagat cgattcagtc 5700
tgcagtatgg actatggagt aaactgcatg ctgaaacccg aaccactgct gaaactgcat 5760
gctgaaaccc gaaccactgc tacggcagtt gccaggatag caggagggcc tttatgcaca 5820
gtggaattga gtag 5834
<210> 57
<211> 471
<212> DNA
<213> Artificial Sequence
<220>
<223> Zea mays - cDNA coding for CENH3
<400> 57
atggctcgaa ccaagcacca ggccgtgagg aagacggcgg agaagcccaa gaagaagctc 60
cagttcgagc gctcaggtgg tgcgagtacc tcggcgacgc cggaaagggc tgctgggacc 120
gggggaagag cggcgtctgg aggtgactca gttaagaaga cgaaaccacg ccaccgctgg 180
cggccaggga ctgtagcgct gcgggagatc aggaagtacc agaagtccac tgaaccgctc 240
atcccctttg cgcctttcgt ccgtgtggtg agggagttaa ccaatttcgt aacaaacggg 300
aaagtagagc gctataccgc agaagccctc cttgcgctgc aagaggcagc agaattccac 360
ttgatagaac tgtttgaaat ggcgaatctg tgtgccatcc atgccaagcg tgtcacaatc 420
atgcaaaagg acatacaact tgcaaggcgt atcggaggaa ggcgttgggc a 471
<210> 58
<211> 157
<212> PRT
<213> Zea mays
<400> 58
Met Ala Arg Thr Lys His Gln Ala Val Arg Lys Thr Ala Glu Lys Pro
1 5 10 15
Lys Lys Lys Leu Gln Phe Glu Arg Ser Gly Gly Ala Ser Thr Ser Ala
20 25 30
Thr Pro Glu Arg Ala Ala Gly Thr Gly Gly Arg Ala Ala Ser Gly Gly
35 40 45
Asp Ser Val Lys Lys Thr Lys Pro Arg His Arg Trp Arg Pro Gly Thr
50 55 60
Val Ala Leu Arg Glu Ile Arg Lys Tyr Gln Lys Ser Thr Glu Pro Leu
65 70 75 80
Ile Pro Phe Ala Pro Phe Val Arg Val Val Arg Glu Leu Thr Asn Phe
85 90 95
Val Thr Asn Gly Lys Val Glu Arg Tyr Thr Ala Glu Ala Leu Leu Ala
100 105 110
Leu Gln Glu Ala Ala Glu Phe His Leu Ile Glu Leu Phe Glu Met Ala
115 120 125
Asn Leu Cys Ala Ile His Ala Lys Arg Val Thr Ile Met Gln Lys Asp
130 135 140
Ile Gln Leu Ala Arg Arg Ile Gly Gly Arg Arg Trp Ala
145 150 155
<210> 59
<211> 8441
<212> DNA
<213> Beta vulgaris
<220>
<221> Intron
<222> (99 )..(203)
<220>
<221> Intron
<222> (245 )..(331)
<220>
<221> Intron
<222> (344)..(488)
<220>
<221> Intron
<222> (591)..(3263)
<220>
<221> Intron
<222> (3339)..(4377)
<220>
<221> Intron
<222> (4458)..(8130)
<400> 59
ctactctttc tctctctctc tctctccatt tctgtttgaa atcatgagag ttaaacacac 60
tgctgccagg aaatcaacca ccaacggtcc tcgttcaagt tagtttcctc tctcttcttc 120
ttttttgttc gcattctctc aatctatatt tcaaatttga aaaaaattgt gatgctcata 180
aaccctaaaa ttttcttgta cagaggctca gaaatctccg cgcagtttgc aatcaccaca 240
atcggttctc tctttgtact tttgatttgt ttttccttca tttgttcgat gaatggctct 300
taattgtctt ttatttactt gaaaattgca gccttctagt agttcaaagc gcaaatcact 360
cagaaacact gatgcaactc ctcaaagtaa ctttttcttt aatattaggt ttaattttac 420
tgctgtttgc caaattctgt tgaaattgta aaatattttt tttcttaaat ttgacggttt 480
cagagaagaa ggcttaccgc cgtaagccgg gcactgtggc actctgggaa atacgcaaat 540
ttcagaagtc attcaagccc ttgattcctg ctgcgccttt cattcgaaca gtatgtattt 600
tttttgtttg tacttaataa atgaattttg gactggtgtt tgtgtggctg catagaaata 660
tatttccata caactgaaat tgtcctagga ggtatcgatg aatgtttgct acaaaataaa 720
taaatataag tgattatatc ttgttaaaaa gccattataa ttgcaactta tatgtatgtt 780
gtaatgaggt caactagcta ttttgtgcaa agtcacccac actttaacat aattttgtgc 840
tctcgtaacc ttaaaaaaat ataagtaaag ggttgatttg gtctaattag agctgatgaa 900
acccaattag attgaaacat aaggtgaaat caggtggtga tcagcttcaa ttagatctaa 960
taagtgcagt ttagtttagc ttcggtgaaa tgaacacacc cttaaagata gaaaatcgac 1020
actatatatg gtccttttta gatatgatag ttcgatattc tgttttgggg tgtgttgaat 1080
gattaaatgg agtggtgaat agctgatggg aactagagaa gatgctcagt agacagttat 1140
tgtggagact atattactga ttacccctgt ttctgagtgg ttaggacaat gtgacaattg 1200
attttgggta ttatttgtag atgtttttct ttttgttaaa agtgccaaga taggtgtgca 1260
gttgctgatt ctcagtttgc taagaattag ctgtgtctgt atttcgtacc tcagttgatt 1320
ctaagtgaac atttctttga attgatgctt tgttcttgca tcatgcaact tggtgaagct 1380
ttcttgtagt tgctccagtg gcaatctagt ctggtatgtt tagaactctt gtgatggtat 1440
gagttcatca agatattggt gatccaatta gcctaaccaa tgttttttac cccctattgt 1500
cactgactta tactccctta tctataaaaa taattgtgac attgatccat ctcctcacaa 1560
tcattaatat tatatgtact gaccatcttt acactctcaa cactgaatct aagtagggga 1620
attttgggaa attcaatgat gaactagtac acccttcttc ccaataatat tgttgacctt 1680
tttattttga tttgtcccat attgtcctct ttggtaattt aatgtatatt cacccaattt 1740
tcttttcaat acccactata ccaacatata attggttaat tcatttttat taattatatt 1800
tcctaagagc ttgttgtgta aacgtggatg aatttgtagg catggatgaa gtattgttat 1860
aatgaggtga caacattact taatttcgaa ctgagggaca gagggatatg atgataaaac 1920
aacttttgct tgcttcttaa actcagaaga tagggtttac accaagtggc atgtaaaagt 1980
cactagatga ttatctatta caagggcttg tacaatctga agtacgatag gatttgaagt 2040
taacaacatt catcgaaagc tcataacttg tccttatatc aatataagtt gctggcatgt 2100
gaaattgcgt tgcaagcatc catgagctag ctcaactatt aactattaaa ctttatattt 2160
ttgcttgatc tagtatgagt cctactattt agtttctcca tctaccttaa tatgtcgcat 2220
acaccaacta atcattatcg ctagaatcaa taaacaaagc tttctttcct taggtgtatt 2280
agtacctagc tcctgtaata ccaagagcac ccaaattggg aagaaaaagt agaattggct 2340
catatctcta atcctacatt gatcattgaa aaggacctta aggttctcat actgaaacat 2400
catctttttg agcaggatat ctacgtagac gacaagaaag actactttgg ttgcccgtgc 2460
atttgagtgc atcagacaac ttctttacca ctgtctaacg gcttgctttg gccatattgt 2520
ggtcttctat gccaaaatta atgatatttc ttggcaccgc gctaatgata ttactgaatg 2580
cggatatcgt acgattagaa tttattcaaa gtaggtagca attactagtt ttgagcattg 2640
agtttcaata attagtaaat taagtgctaa acttgtacat tttggctaca tgtatttgaa 2700
ttagaattgg tacgaggaaa tatagcaaca ttacgggcaa tgttcactca agtagaagcc 2760
attacatcaa atagtactag ttgaagtatt agttctcata atactaatca ttgtcattaa 2820
tggaatattg gaacgtaaat gcctttaagg tgctgtagtt ttagtagaaa ttctactatt 2880
ctagtatgat aatgcaattt attgaaactg tttgtaagat agcttggatc ccacatcagt 2940
cttgatgcta aataaatgga tgtccataat cttctaatct ttaatttgtg tctcttacca 3000
aacgagaaaa aataggagaa atccaatttg catgacctca ataaggaaat gttgttaatg 3060
tgtgatgctt gtttctcatt tatagtctag agagagttat catgtccaag attgcagtct 3120
tggtactgag aaagtttgat tgttggttgg ctgcttcttg agcctctttt ttttagagta 3180
agacacttcc tagatataat tttctttatt tttttgtaaa ttccatatat actactacat 3240
taacaggttt aagtttatat taggtgagag agattactca ccagtttgct ccttatgttg 3300
gtcgttggca agctgaagct ctgatggccc ttcaagaggt gcagaccaac tcttttagcc 3360
tttttttttc tggcatgtca agtgtggcta ttagattttc tgtgtgattc tcactcccat 3420
atatctatat atgtacatat taaagcacat tgatacctat cttgtcagat gtggtctttt 3480
caattctttt ctaagttgag attcttctct tggtcgtaga tatgctcctg ccgaaatata 3540
ctgctgtctt gttatccatc atgacttggt tatgcttgta tctgggcatt atcttggcat 3600
gcttaaaaac aagtattgaa cgagcctcct attgataaat tttactatta atattggatg 3660
gcttctcaaa ttctaatggc agtgagatac tgttaagttg ggagaaatag attaagaaac 3720
agaaagatgt ttaccatgag agcaattgaa atagaaaata gagtaacttt ttgcaaagat 3780
tttggtcctt tagattgttg aatactacct gataatgaag cattttctaa atttatgtgc 3840
tttctatcta tcagatactg gaatacaatc aaattcctat cacgtactga gcattgtgat 3900
cagattcttg cttgcttcct atcacatact ggaatccttt tgattgttga atacaaagat 3960
aatgaagcat tctctaactt tatgtgctgt aactacttat aatgattctt gcttgcctcc 4020
tatcacgtac tcaaatcctt tgtttgattt gtctcttata agaggaactt cctgtctttc 4080
tttgtcatga cttagtattt atagaggtgc caacttatgg ccttgacaac tgaagctttt 4140
atgcaaactc cggattttgt tgatggaagt acaagtaaca ctttagcatg tggattcagg 4200
tctaacggtt aagacttttt aatgaatgtt ttaactgtag tagtttattg atataaaaaa 4260
agtggtctct caaacttttt atgagatcat atcgaagtaa tcaaatttat gattcaggtg 4320
cttctgctat tattcttggt taagcatgtg ctatttttga cagtctgtca attgtaggct 4380
gcagagaatt ttattgtccg tttgtttgaa gatggtatgc tttgtgcaat tcatgccaaa 4440
cgagttacac tcagtgagta tctgatttcc ttcggtggtg ctgctattat gcattatata 4500
cactttgcct caatatcgtt atataaggag tccttgtttt catatttgtt tgatgcatat 4560
gttatatcct gtttagtggc tgctgcagtt gtgaacttac ggcctgtttg attagtggtc 4620
ataaatgatg gtaatactaa tataatttag tataaatttg taaaaaaaat gctaatatca 4680
atatttatgg taatgaaatt ttatcataaa acatgagttc tcttttataa gttttcatta 4740
ctatccaata ccaccttccc aagtggtaat gaacggtaat gaaattttag gaagaaaatg 4800
gatatttggg gattagatag cattaccatg ggtaatgaca tgagattttc tttacaactt 4860
tatactacga tgcattatca ttaccaccat ttatgaccca taaccaaaaa aaccataatg 4920
tgttaggttc atttttcatt tttctaataa tttgcttcat gaattttttc tggagatatc 4980
ttatctagat atttcttgcc aacatgtttc acctgataat tgatcgattt aatagttcag 5040
aactttccaa aaactatgct gctcggtgtt ggctgtcatc catcagttta agaaaactat 5100
tgacatgatt taagcctcgt cctgtactac taggaagggt aaactattgt tgcttccaaa 5160
aatgtctttt aagggcgtgt tcagcaacaa tagttgtagt agtagctttt agctgttagt 5220
tgtgctcgta gctgttagtg gttagtgtgt aactgttagc tgttcaagta gcggtataag 5280
atattgatgt tcggtaaaag aagctgtcaa aatagctgtt tacaaagaat taataaaaaa 5340
ctcaaacaaa gctttaatat ataatttatg caccactaaa gctaccccaa aagctacaaa 5400
ttgtagcttt ttacaaacac tactaaaaca ctacttgtaa cactaaaagc tacttatact 5460
actattttgc caaacattat tattttttct taattagtgt tttgacctag tcaagacact 5520
aaaagctact tgaaaagctt ttgccgaaca cgcccttagt agacaagagg ggggaggggg 5580
tcatcaagaa aatatgatta tactctcaac aaaaaaaaaa tgtaacttaa aaaaaataaa 5640
aataaataat tgactacttc aattaagaaa agaatagaat aaaaacatta cagtggatgt 5700
ctcatccaca tccctaattt aatggcacaa tagaataatt gttttaaatt ttagaaatta 5760
caacacaaga tgtaaattac tcttatcttc ctcttcgtaa tctttttact cttcctttac 5820
ctcttccttt acctctacat aaaatagaga attagagatt gattaagata attataagat 5880
tttagaaaca ttggttaaga aattcttcaa caaacataat caagtaactc cattatttta 5940
gtttagtgac ttgctattta tcaccctaat ttcaccatct accgccctcc ttggacaata 6000
ttgccccttc cactttcttc actcttcctt cctcacgcat cttatcatct ccttccacta 6060
tcacctttaa aaaagtgtgt caggcacaac aaaaacgctt ttatcaaccc acgcgaggcg 6120
aagtacgtca ggcgcaacaa ggcgcgcacc taattctgtc ttttgccaag gctgatggtg 6180
cacttagttt taaaaagcgc agcaaagatg tgcctaggcg caaggcggtg aaaaaattgc 6240
atccgtcagc agcggagtag aggctcacaa caataggtgc gaagaggcgt gcacgtacaa 6300
aagaagcaaa aataagaaac tcaaatatga gacccagtgt ttaacatgta aattcgatac 6360
ccagtgttta acatgtaaat tcgattaaaa gcccttaatt aattgcatga aattaattca 6420
ttttaaccta tactaaaagc cctaatatta gaaaatccta gtttgcaggt tgaggaattt 6480
ggaaaattga tgattgttgg atttgaaaaa attgttgccg gcgatgaatg tgaggtggtt 6540
tatggcacta gagaggttgg cgttcgttgc cgatgaagct ttccaaggtc attctctcct 6600
tgtcttcttc ctatgcctag ctctcttccc tctccttaat cttctcttct tttctattct 6660
ctctctttat cactacatta tgtttatttc tcgttcttcc cctatgtctt tcacttggac 6720
acttcggggg tatcttcatc ttttatctgc aatttgaagt ttgagaagct tccagagtcg 6780
agtgttaaac ttttgcttct ttttttttaa tcttttgccc ttttttctta gtggcccttg 6840
actagtgatg cacatgtgac caattactaa atgagctttt attttgtctc tcttcttttt 6900
caagcaattt tttttaagta aatcatctaa aacaaagtac tatccatttt agttgtgtaa 6960
atggtgctat tttaaaaccg cacaaaaatt aaaaacataa aaataaaggt gtgcttcgca 7020
tacaagatgt atgcgccttc gtcttgcccc ttttgagact aagactacca taagaactta 7080
gtcacttgag aatggaatgg gtgcaagatg gacgacgata attctaaaga cctctagaag 7140
gatagtgtat agtaactaat acgaaccgaa atataagttt aactaaaatt ttaaagtcta 7200
tatttccata tggtatatgc tggaatacac gaaatgtcca gaatttgtag tggaccacga 7260
tccacacgtc ttttcaggat tctaggtgta ttccaacgaa aaatataaga aaaccatatt 7320
ctactatctg gttgttgtca tcctttcctt gccggcgtga cttctcatcc ttttattttt 7380
gtccggtgct ggtgacacac tttcctatga tagtgtggtg caaagtaagg tgatgatatg 7440
gtgttttgta gaggtgtggt gatttttgtg gtggtgggtg gaagaggggt ggttgcatat 7500
agaaaggggt aagagtcaat gaggggtgga aggggacaag gggtatattg gtaaatgcat 7560
gtaacattag ggtggtgttg agtaattttt gggaagttaa tataaactac ccccttttgg 7620
tacaagagag aatacccgaa ctactgctct gatatttttg ttcacgttat ttgatgtaat 7680
tacgcaatta atttgttttc tataagcttc cgcacacaat tgtgcatata aggctagtct 7740
aatatgagac accaacataa ctgactttct tttgcaacga aggtaccttg tcagatttag 7800
aacatagcat caggatttta tttgttgtat ctgtcatcct tgtttattgc tttaattatg 7860
ctttgtatga tgcattttac cacttcgtat gaaaaaaagt gaaatttcat ttagtggtca 7920
tttacatatt acgagttgtg gacatgtttg aacatttgat tttggaaatt ttaagcctca 7980
tattatggag atttattgga cacaaatata gccataattc tccatcaact tgtttctaga 8040
agtgttgctc ttcctgatgt acttgaattc taattaggtt ttatcagaca ttatattata 8100
atgatatgat ttacaatttg ttgtagtgaa aaaggatttg gagctcgcgc gaaggattgg 8160
gggcagagag aggggatggt aactaaacaa cacagatgac tcatttattt aagggccaac 8220
aattgaattc gctgttgatt tcatctgtat atactgctct aggcttctat tccaatgtaa 8280
tttataaatc caaggttagt agcatgttaa gctttgtatt cagtataatg agacttatat 8340
tttgcagttg agattttagt tgtttgatgt gacttgtaaa ttgtaacttg taagtgacgt 8400
cttgaggatt atcgggacaa tatactattt tttttttcaa a 8441
<210> 60
<211> 462
<212> DNA
<213> Artificial Sequence
<220>
<223> Sorghum bicolor - cDNA coding for CENH3
<400> 60
atgagagtta aacacactgc tgccaggaaa tcaaccacca atggtcctcg ttcaaaggct 60
cagaaatctc cgcgcagttt gcaatcacca caatcgcctt ctagtagttc aaagcgcaaa 120
tcacgcagaa acactgatgc aactcctcaa aagaagaagg cttaccgccg taagccgggc 180
actgtggcac tctgggaaat acgcaaattt cagaagtcat tcaagccctt gattcctgct 240
gcgcctttca ttcgaacagt gagagagatt actcaccagt ttgctcctta tgttggtcgt 300
tggcaagctg aagctctgat ggcccttcaa gaggctgcag agaattttat tgtccgtttg 360
tttgaagatg gtatgctttg tgcaattcat gccaaacgag ttacactcat gaaaaaggat 420
ttggagctcg cgcgaaggat tgggggcaga gagaggggat gg 462
<210> 61
<211> 154
<212> PRT
<213> Beta vulgaris
<400> 61
Met Arg Val Lys His Thr Ala Ala Arg Lys Ser Thr Thr Asn Gly Pro
1 5 10 15
Arg Ser Lys Ala Gln Lys Ser Pro Arg Ser Leu Gln Ser Pro Gln Ser
20 25 30
Pro Ser Ser Ser Ser Lys Arg Lys Ser Arg Arg Asn Thr Asp Ala Thr
35 40 45
Pro Gln Lys Lys Lys Ala Tyr Arg Arg Lys Pro Gly Thr Val Ala Leu
50 55 60
Trp Glu Ile Arg Lys Phe Gln Lys Ser Phe Lys Pro Leu Ile Pro Ala
65 70 75 80
Ala Pro Phe Ile Arg Thr Val Arg Glu Ile Thr His Gln Phe Ala Pro
85 90 95
Tyr Val Gly Arg Trp Gln Ala Glu Ala Leu Met Ala Leu Gln Glu Ala
100 105 110
Ala Glu Asn Phe Ile Val Arg Leu Phe Glu Asp Gly Met Leu Cys Ala
115 120 125
Ile His Ala Lys Arg Val Thr Leu Met Lys Lys Asp Leu Glu Leu Ala
130 135 140
Arg Arg Ile Gly Gly Arg Glu Arg Gly Trp
145 150
<210> 62
<211> 4001
<212> DNA
<213> Brassica napus
<400> 62
tgtccgggag gatccaccgg cggtggtcgt tactccattt taacttgatg tcttgaaagc 60
agaggacatg gtatggtggc ggcagaatct atttttagtt gttaattttt cttttcctct 120
gattcttttt atttttttcg aatgaactaa ctttgggttt attcagaaga attatcatct 180
aaaaactgat tcaataaaca aaataattta catatttcac aatgagccat tagtaaacaa 240
gtcgaaagtg aaaccaaatg ggaagagaac aattttaata aaaatatgtt ctaatttcct 300
actttttatg aattgaactc ccgaagagaa tggccgaaga acggagtaaa agctcaatga 360
ttgtaaaagc tatgtattct cttgctttga ggaaaaagct ttttgtttgc actcataggc 420
ctgatatgtt gtgatggctc tttacatatt gggtcttttg tggtctatta aacggttact 480
gaagaattag tttatcgcat ttaaaaaaaa atatttgaaa accacatatc taaaatctca 540
atatattatt tattgataga tgaaaagaaa aatagtttat aaaataatat taattggtaa 600
atggaagtca aaattttaca tttaatacat gactagttcg attctccggc tgaaacccat 660
ttaccactga gccaagacca cttgattata tatatcaagc tcttgttttt tgcattaaca 720
aaagttgcat ccaaaatttc aaaacaacaa ctaaattaat gtctttctat tttcaaagtt 780
ttatctccaa acctattaat agtttaattt ttttttttaa aattaggaaa ccggttaaaa 840
tatattttaa atacaaaaaa cttaaacaat gaatcattta tttatttatt cttcaaaatt 900
taaatatccg aacccggccc aaaatatccg aacccgaaca taaaatatcc gaacccgact 960
cgaagtgtag aaaatatccg aacgggtttt atacctttat actgaaatac cctatacgaa 1020
cccgaatgtg tatccgaacg ccccctacaa tatatgatca tcatttgtat cttgattgaa 1080
caaaaaaaaa gttaaactat tgatcacaaa attttcaatg tgagactttt accattttta 1140
gtcatttata gtcgttttta aaaattcaaa atataactta taagaaaaaa tctaattttt 1200
tttattatat gcttaatgtg attgtttaat ttcttttaat aatataaaat taaacaaaaa 1260
atgagaggtt aaaaaaattg ttatcaaata tgtattattc ataatcatta attgtcatat 1320
atatgttaat tatattaggt aatttcgtag tttttattta agaaaagaaa aaaatattat 1380
tttgtacact actaattaat ttgatagtta gtttaataaa aaatatatta tattattata 1440
tggaccaact tatttttcta aaaaaaaacc actgttttaa aaaccaaacc aactataaac 1500
cggagatata ccggattgag tggctaaaac actctttgta tatatgtgct gagcaaaccc 1560
tctgagtgag atggcgtgtt aagaagtagg aggaccattc atgcctctta tgagttgtag 1620
tctgtgtgta caaaaaagaa gcgttggtgt gaaagaaagc agaaggattt gaaaatcaaa 1680
aaaattgaag gagaagcggg aaaacaaata atctctccct ccgctttttt ttctccaaat 1740
aatcaatctc tcatttcatt tgttaaccca agtttttgat aattatttca aaggggttta 1800
tttatctttt attcctccgg cggcagtaag tagtaatcaa tggcgagaac caaacatttc 1860
gcttccaggg cacgagatcg caatcgaact agttagtact ctctctctct ctgccttttt 1920
tttgatattt attttctagg ttaaacccta atttggcatc tgaaatttgt agatgcgact 1980
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcgg tacgtcatct attttctttt 2040
cccgttttag gtttttacgc aaatctcgtt actgtttttt tgacgaatcg attgaaatgt 2100
gtagaccccg acgagaagag aaggcagcca aggagaagct caacagagtg agtctttcta 2160
tttcattttc tgagatccat gaatcctttt catctctcgt gtgttgtgac atgaatcaat 2220
tgcagcagca actcctacta cgactccacc agccggtaga aaagtaagtt acatttccat 2280
ttcacaccat tcatttgctt ctttatcaac aaactgctct ctcatctgtt ttttttgttt 2340
tgttttggtt ttgtgaagaa aggagggact aagcgaacta aacaagctat gcctaaaagt 2400
tagtgacaga ttttaaaatc tctattttgg atcatcattc tctcaggaca tgtctatttg 2460
catttgttct tattatgtct gtctgtctgt ctttgtcccc cttgtaggtt ccaacaagaa 2520
gaagacattc cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa 2580
gaccaccaaa cttcttatcc ctgccgctag tttcatccga gaagttagta atgaactttg 2640
ttattcatac attcccgctt acttgttttc aatgactctg caattactga tatagaattt 2700
ggagcaacca ttatggggtg atttctctaa ctacaaatta ctaatactat cccaggtgag 2760
aagtgtcacc cagatctttg cctctcccga tgttacccgt tggactgctg aagctcttat 2820
ggctattcaa gaggtacgtg tactccttcc ctcttttgtt tcctattttc cacttgatgt 2880
ctaatttaaa ctgatcgttt tttttttata tttcttttgg tgtggggcgg ggcaggcggc 2940
tgaagatttt ttaattggct tgttctctga tgctatgctt tgcgctatcc atgcaaggcg 3000
tgttactcta agtaagtagt actccccaaa ataaggaaac ccattttata tacaacattg 3060
cctcatccat gtctgcttct cttcatatca gtgagaaaag attttgagct tgcacgccgt 3120
cttggaggaa aaggcagacc attgtgatcg tttcgcaggt tgtataactt tgttcactcc 3180
ttatgtcttg tcatttgtga tctgactgac actttctttt gaaacataac tgcttgattc 3240
aatatctagg ctgtaaaact tatccctcct tgtttactat cttatatgct ttttccttgg 3300
aattgatagt ttccattgag atttcacttg cacgaaacat atctgctttc tcaatatctc 3360
tcagtcttag aaagggctat tgactaaaag aaaagaaaat ttagaggaag atttgtaaag 3420
acatgtgttt agagagggct taattaaaaa cacacgcttc tgctagcctt gctatttgat 3480
tcccaatttc aacttttttc gaggcatatt ataaagtttt taaatgtact tggcacttca 3540
acttttataa tttatataac gattttattc taatagagca tttgtgattt catagtgttg 3600
tcatgaaact caagtaattc acaccgtccg atgttgctat tgtctaataa aatgttgaaa 3660
aaattgtcaa aacagaacaa aaaacaacat agttgtctct atggtataaa actatcacta 3720
agttgtctct atagtataat atttttcgca atcccaaaac taatttttct ttaatcaaat 3780
taaacataaa ctaaaaccat ttttaaaaag tttaatggaa aaagataaaa aaataaggta 3840
atctcgtaat gttttaaaaa ggaaaaaaaa tgtaaaaaca atttaaaaaa aagaacacac 3900
gacacagatc aaaaatatca tgtaatctaa ttgcatttgg tttctaaaat cttccaaaac 3960
tattctttta aaattctcta aggtaaaact tgattccaat a 4001
<210> 63
<211> 540
<212> DNA
<213> Brassica napus
<400> 63
atggcgagaa ccaaacattt cgcttccagg gcacgagatc gcaatcgaac taatgcgact 60
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcga ccccgacgag aagagaaggc 120
agccaaggag aagctcaaca gacaactcct actacgactc caccagccgg tagaaaaaaa 180
ggagggacta agcgaactaa acaagctatg cctaaaagtt ccaacaagaa gaagacattc 240
cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa gaccaccaaa 300
cttcttatcc ctgccgctag tttcatccga gaagtgagaa gtgtcaccca gatctttgcc 360
tctcccgatg ttacccgttg gactgctgaa gctcttatgg ctattcaaga ggcggctgaa 420
gattttttaa ttggcttgtt ctctgatgct atgctttgcg ctatccatgc aaggcgtgtt 480
actctaatga gaaaagattt tgagcttgca cgccgtcttg gaggaaaagg cagaccattg 540
<210> 64
<211> 180
<212> PRT
<213> Brassica napus
<400> 64
Met Ala Arg Thr Lys His Phe Ala Ser Arg Ala Arg Asp Arg Asn Arg
1 5 10 15
Thr Asn Ala Thr Ala Ser Ser Ser Ala Ala Ala Ala Glu Gly Pro Ser
20 25 30
Ala Thr Pro Thr Arg Arg Glu Gly Ser Gln Gly Glu Ala Gln Gln Thr
35 40 45
Thr Pro Thr Thr Thr Pro Pro Ala Gly Arg Lys Lys Gly Gly Thr Lys
50 55 60
Arg Thr Lys Gln Ala Met Pro Lys Ser Ser Asn Lys Lys Lys Thr Phe
65 70 75 80
Arg Tyr Lys Pro Gly Thr Val Ala Leu Arg Glu Ile Arg His Phe Gln
85 90 95
Lys Thr Thr Lys Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val
100 105 110
Arg Ser Val Thr Gln Ile Phe Ala Ser Pro Asp Val Thr Arg Trp Thr
115 120 125
Ala Glu Ala Leu Met Ala Ile Gln Glu Ala Ala Glu Asp Phe Leu Ile
130 135 140
Gly Leu Phe Ser Asp Ala Met Leu Cys Ala Ile His Ala Arg Arg Val
145 150 155 160
Thr Leu Met Arg Lys Asp Phe Glu Leu Ala Arg Arg Leu Gly Gly Lys
165 170 175
Gly Arg Pro Leu
180
<210> 65
<211> 4001
<212> DNA
<213> Brassica napus
<400> 65
tgtccgggag gatccaccgg cggtggtcgt tactccattt taacttgatg tcttgaaagc 60
agaggacatg gtatggtggc ggcagaatct atttttagtt gttaattttt cttttcctct 120
gattcttttt atttttttcg aatgaactaa ctttgggttt attcagaaga attatcatct 180
aaaaactgat tcaataaaca aaataattta catatttcac aatgagccat tagtaaacaa 240
gtcgaaagtg aaaccaaatg ggaagagaac aattttaata aaaatatgtt ctaatttcct 300
actttttatg aattgaactc ccgaagagaa tggccgaaga acggagtaaa agctcaatga 360
ttgtaaaagc tatgtattct cttgctttga ggaaaaagct ttttgtttgc actcataggc 420
ctgatatgtt gtgatggctc tttacatatt gggtcttttg tggtctatta aacggttact 480
gaagaattag tttatcgcat ttaaaaaaaa atatttgaaa accacatatc taaaatctca 540
atatattatt tattgataga tgaaaagaaa aatagtttat aaaataatat taattggtaa 600
atggaagtca aaattttaca tttaatacat gactagttcg attctccggc tgaaacccat 660
ttaccactga gccaagacca cttgattata tatatcaagc tcttgttttt tgcattaaca 720
aaagttgcat ccaaaatttc aaaacaacaa ctaaattaat gtctttctat tttcaaagtt 780
ttatctccaa acctattaat agtttaattt ttttttttaa aattaggaaa ccggttaaaa 840
tatattttaa atacaaaaaa cttaaacaat gaatcattta tttatttatt cttcaaaatt 900
taaatatccg aacccggccc aaaatatccg aacccgaaca taaaatatcc gaacccgact 960
cgaagtgtag aaaatatccg aacgggtttt atacctttat actgaaatac cctatacgaa 1020
cccgaatgtg tatccgaacg ccccctacaa tatatgatca tcatttgtat cttgattgaa 1080
caaaaaaaaa gttaaactat tgatcacaaa attttcaatg tgagactttt accattttta 1140
gtcatttata gtcgttttta aaaattcaaa atataactta taagaaaaaa tctaattttt 1200
tttattatat gcttaatgtg attgtttaat ttcttttaat aatataaaat taaacaaaaa 1260
atgagaggtt aaaaaaattg ttatcaaata tgtattattc ataatcatta attgtcatat 1320
atatgttaat tatattaggt aatttcgtag tttttattta agaaaagaaa aaaatattat 1380
tttgtacact actaattaat ttgatagtta gtttaataaa aaatatatta tattattata 1440
tggaccaact tatttttcta aaaaaaaacc actgttttaa aaaccaaacc aactataaac 1500
cggagatata ccggattgag tggctaaaac actctttgta tatatgtgct gagcaaaccc 1560
tctgagtgag atggcgtgtt aagaagtagg aggaccattc atgcctctta tgagttgtag 1620
tctgtgtgta caaaaaagaa gcgttggtgt gaaagaaagc agaaggattt gaaaatcaaa 1680
aaaattgaag gagaagcggg aaaacaaata atctctccct ccgctttttt ttctccaaat 1740
aatcaatctc tcatttcatt tgttaaccca agtttttgat aattatttca aaggggttta 1800
tttatctttt attcctccgg cggcagtaag tagtaatcaa tggcgagaac caaacatttc 1860
gcttccaggg cacgagatcg caatcgaact agttagtact ctctctctct ctgccttttt 1920
tttgatattt attttctagg ttaaacccta atttggcatc tgaaatttgt agatgcgact 1980
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcgg tacgtcatct attttctttt 2040
cccgttttag gtttttacgc aaatctcgtt actgtttttt tgacgaatcg attgaaatgt 2100
gtagaccccg acgagaagag aaggcagcca aggagaagct caacagagtg agtctttcta 2160
tttcattttc tgagatccat gaatcctttt catctctcgt gtgttgtgac atgaatcaat 2220
tgcagcagca actcctacta cgactccacc agccggtaga aaagtaagtt acatttccat 2280
ttcacaccat tcatttgctt ctttatcaac aaactgctct ctcatctgtt ttttttgttt 2340
tgttttggtt ttgtgaagaa aggagggact aagcgaacta aacaagctat gcctaaaagt 2400
tagtgacaga ttttaaaatc tctattttgg atcatcattc tctcaggaca tgtctatttg 2460
catttgttct tattatgtct gtctgtctgt ctttgtcccc cttgtaggtt ccaacaagaa 2520
gaagacattc cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa 2580
gaccaccaaa cttcttatcc ctgccgctag tttcatccga gaagttagta atgaactttg 2640
ttattcatac attcccgctt acttgttttc aatgactctg caattactga tatagaattt 2700
ggagcaacca ttatggggtg atttctctaa ctacaaatta ctaatactat cccaggtgag 2760
aagtgtcacc cagatctttg cccctcccga tgttacccgt tgaactgctg aagctcttat 2820
ggctattcaa gaggtacgtg tactccttcc ctcttttgtt tcctattttc cacttgatgt 2880
ctaatttaaa ctgatcgttt tttttttata tttcttttgg tgtggggcgg ggcaggcggc 2940
tgaagatttt ttaattggct tgttctctga tgctatgctt tgcgctatcc atgcaaggcg 3000
tgttactcta agtaagtagt actccccaaa ataaggaaac ccattttata tacaacattg 3060
cctcatccat gtctgcttct cttcatatca gtgagaaaag attttgagct tgcacgccgt 3120
cttggaggaa aaggcagacc attgtgatcg tttcgcaggt tgtataactt tgttcactcc 3180
ttatgtcttg tcatttgtga tctgactgac actttctttt gaaacataac tgcttgattc 3240
aatatctagg ctgtaaaact tatccctcct tgtttactat cttatatgct ttttccttgg 3300
aattgatagt ttccattgag atttcacttg cacgaaacat atctgctttc tcaatatctc 3360
tcagtcttag aaagggctat tgactaaaag aaaagaaaat ttagaggaag atttgtaaag 3420
acatgtgttt agagagggct taattaaaaa cacacgcttc tgctagcctt gctatttgat 3480
tcccaatttc aacttttttc gaggcatatt ataaagtttt taaatgtact tggcacttca 3540
acttttataa tttatataac gattttattc taatagagca tttgtgattt catagtgttg 3600
tcatgaaact caagtaattc acaccgtccg atgttgctat tgtctaataa aatgttgaaa 3660
aaattgtcaa aacagaacaa aaaacaacat agttgtctct atggtataaa actatcacta 3720
agttgtctct atagtataat atttttcgca atcccaaaac taatttttct ttaatcaaat 3780
taaacataaa ctaaaaccat ttttaaaaag tttaatggaa aaagataaaa aaataaggta 3840
atctcgtaat gttttaaaaa ggaaaaaaaa tgtaaaaaca atttaaaaaa aagaacacac 3900
gacacagatc aaaaatatca tgtaatctaa ttgcatttgg tttctaaaat cttccaaaac 3960
tattctttta aaattctcta aggtaaaact tgattccaat a 4001
<210> 66
<211> 540
<212> DNA
<213> Brassica napus
<400> 66
atggcgagaa ccaaacattt cgcttccagg gcacgagatc gcaatcgaac taatgcgact 60
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcga ccccgacgag aagagaaggc 120
agccaaggag aagctcaaca gacaactcct actacgactc caccagccgg tagaaaaaaa 180
ggagggacta agcgaactaa acaagctatg cctaaaagtt ccaacaagaa gaagacattc 240
cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa gaccaccaaa 300
cttcttatcc ctgccgctag tttcatccga gaagtgagaa gtgtcaccca gatctttgcc 360
cctcccgatg ttacccgttg aactgctgaa gctcttatgg ctattcaaga ggcggctgaa 420
gattttttaa ttggcttgtt ctctgatgct atgctttgcg ctatccatgc aaggcgtgtt 480
actctaatga gaaaagattt tgagcttgca cgccgtcttg gaggaaaagg cagaccattg 540
<210> 67
<211> 126
<212> PRT
<213> Brassica napus
<400> 67
Met Ala Arg Thr Lys His Phe Ala Ser Arg Ala Arg Asp Arg Asn Arg
1 5 10 15
Thr Asn Ala Thr Ala Ser Ser Ser Ala Ala Ala Ala Glu Gly Pro Ser
20 25 30
Ala Thr Pro Thr Arg Arg Glu Gly Ser Gln Gly Glu Ala Gln Gln Thr
35 40 45
Thr Pro Thr Thr Thr Pro Pro Ala Gly Arg Lys Lys Gly Gly Thr Lys
50 55 60
Arg Thr Lys Gln Ala Met Pro Lys Ser Ser Asn Lys Lys Lys Thr Phe
65 70 75 80
Arg Tyr Lys Pro Gly Thr Val Ala Leu Arg Glu Ile Arg His Phe Gln
85 90 95
Lys Thr Thr Lys Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val
100 105 110
Arg Ser Val Thr Gln Ile Phe Ala Pro Pro Asp Val Thr Arg
115 120 125
<210> 68
<211> 4001
<212> DNA
<213> Brassica napus
<400> 68
tgtccgggag gatccaccgg cggtggtcgt tactccattt taacttgatg tcttgaaagc 60
agaggacatg gtatggtggc ggcagaatct atttttagtt gttaattttt cttttcctct 120
gattcttttt atttttttcg aatgaactaa ctttgggttt attcagaaga attatcatct 180
aaaaactgat tcaataaaca aaataattta catatttcac aatgagccat tagtaaacaa 240
gtcgaaagtg aaaccaaatg ggaagagaac aattttaata aaaatatgtt ctaatttcct 300
actttttatg aattgaactc ccgaagagaa tggccgaaga acggagtaaa agctcaatga 360
ttgtaaaagc tatgtattct cttgctttga ggaaaaagct ttttgtttgc actcataggc 420
ctgatatgtt gtgatggctc tttacatatt gggtcttttg tggtctatta aacggttact 480
gaagaattag tttatcgcat ttaaaaaaaa atatttgaaa accacatatc taaaatctca 540
atatattatt tattgataga tgaaaagaaa aatagtttat aaaataatat taattggtaa 600
atggaagtca aaattttaca tttaatacat gactagttcg attctccggc tgaaacccat 660
ttaccactga gccaagacca cttgattata tatatcaagc tcttgttttt tgcattaaca 720
aaagttgcat ccaaaatttc aaaacaacaa ctaaattaat gtctttctat tttcaaagtt 780
ttatctccaa acctattaat agtttaattt ttttttttaa aattaggaaa ccggttaaaa 840
tatattttaa atacaaaaaa cttaaacaat gaatcattta tttatttatt cttcaaaatt 900
taaatatccg aacccggccc aaaatatccg aacccgaaca taaaatatcc gaacccgact 960
cgaagtgtag aaaatatccg aacgggtttt atacctttat actgaaatac cctatacgaa 1020
cccgaatgtg tatccgaacg ccccctacaa tatatgatca tcatttgtat cttgattgaa 1080
caaaaaaaaa gttaaactat tgatcacaaa attttcaatg tgagactttt accattttta 1140
gtcatttata gtcgttttta aaaattcaaa atataactta taagaaaaaa tctaattttt 1200
tttattatat gcttaatgtg attgtttaat ttcttttaat aatataaaat taaacaaaaa 1260
atgagaggtt aaaaaaattg ttatcaaata tgtattattc ataatcatta attgtcatat 1320
atatgttaat tatattaggt aatttcgtag tttttattta agaaaagaaa aaaatattat 1380
tttgtacact actaattaat ttgatagtta gtttaataaa aaatatatta tattattata 1440
tggaccaact tatttttcta aaaaaaaacc actgttttaa aaaccaaacc aactataaac 1500
cggagatata ccggattgag tggctaaaac actctttgta tatatgtgct gagcaaaccc 1560
tctgagtgag atggcgtgtt aagaagtagg aggaccattc atgcctctta tgagttgtag 1620
tctgtgtgta caaaaaagaa gcgttggtgt gaaagaaagc agaaggattt gaaaatcaaa 1680
aaaattgaag gagaagcggg aaaacaaata atctctccct ccgctttttt ttctccaaat 1740
aatcaatctc tcatttcatt tgttaaccca agtttttgat aattatttca aaggggttta 1800
tttatctttt attcctccgg cggcagtaag tagtaatcaa tggcgagaac caaacatttc 1860
gcttccaggg cacgagatcg caatcgaact agttagtact ctctctctct ctgccttttt 1920
tttgatattt attttctagg ttaaacccta atttggcatc tgaaatttgt agatgcgact 1980
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcgg tacgtcatct attttctttt 2040
cccgttttag gtttttacgc aaatctcgtt actgtttttt tgacgaatcg attgaaatgt 2100
gtagaccccg acgagaagag aaggcagcca aggagaagct caacagagtg agtctttcta 2160
tttcattttc tgagatccat gaatcctttt catctctcgt gtgttgtgac atgaatcaat 2220
tgcagcagca actcctacta cgactccacc agccggtaga aaagtaagtt acatttccat 2280
ttcacaccat tcatttgctt ctttatcaac aaactgctct ctcatctgtt ttttttgttt 2340
tgttttggtt ttgtgaagaa aggagggact aagcgaacta aacaagctat gcctaaaagt 2400
tagtgacaga ttttaaaatc tctattttgg atcatcattc tctcaggaca tgtctatttg 2460
catttgttct tattatgtct gtctgtctgt ctttgtcccc cttgtaggtt ccaacaagaa 2520
gaagacattc cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa 2580
gaccaccaaa cttcttatcc ctgccgctag tttcatccga gaagttagta atgaactttg 2640
ttattcatac attcccgctt acttgttttc aatgactctg caattactga tatagaattt 2700
ggagcaacca ttatggggtg atttctctaa ctacaaatta ctaatactat cccaggtgag 2760
aagtgtcacc cagatctttg cccctcccga tgttacccgt tggactgctg aagcttttat 2820
ggctattcaa gaggtacgtg tactccttcc ctcttttgtt tcctattttc cacttgatgt 2880
ctaatttaaa ctgatcgttt tttttttata tttcttttgg tgtggggcgg ggcaggcggc 2940
tgaagatttt ttaattggct tgttctctga tgctatgctt tgcgctatcc atgcaaggcg 3000
tgttactcta agtaagtagt actccccaaa ataaggaaac ccattttata tacaacattg 3060
cctcatccat gtctgcttct cttcatatca gtgagaaaag attttgagct tgcacgccgt 3120
cttggaggaa aaggcagacc attgtgatcg tttcgcaggt tgtataactt tgttcactcc 3180
ttatgtcttg tcatttgtga tctgactgac actttctttt gaaacataac tgcttgattc 3240
aatatctagg ctgtaaaact tatccctcct tgtttactat cttatatgct ttttccttgg 3300
aattgatagt ttccattgag atttcacttg cacgaaacat atctgctttc tcaatatctc 3360
tcagtcttag aaagggctat tgactaaaag aaaagaaaat ttagaggaag atttgtaaag 3420
acatgtgttt agagagggct taattaaaaa cacacgcttc tgctagcctt gctatttgat 3480
tcccaatttc aacttttttc gaggcatatt ataaagtttt taaatgtact tggcacttca 3540
acttttataa tttatataac gattttattc taatagagca tttgtgattt catagtgttg 3600
tcatgaaact caagtaattc acaccgtccg atgttgctat tgtctaataa aatgttgaaa 3660
aaattgtcaa aacagaacaa aaaacaacat agttgtctct atggtataaa actatcacta 3720
agttgtctct atagtataat atttttcgca atcccaaaac taatttttct ttaatcaaat 3780
taaacataaa ctaaaaccat ttttaaaaag tttaatggaa aaagataaaa aaataaggta 3840
atctcgtaat gttttaaaaa ggaaaaaaaa tgtaaaaaca atttaaaaaa aagaacacac 3900
gacacagatc aaaaatatca tgtaatctaa ttgcatttgg tttctaaaat cttccaaaac 3960
tattctttta aaattctcta aggtaaaact tgattccaat a 4001
<210> 69
<211> 540
<212> DNA
<213> Brassica napus
<400> 69
atggcgagaa ccaaacattt cgcttccagg gcacgagatc gcaatcgaac taatgcgact 60
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcga ccccgacgag aagagaaggc 120
agccaaggag aagctcaaca gacaactcct actacgactc caccagccgg tagaaaaaaa 180
ggagggacta agcgaactaa acaagctatg cctaaaagtt ccaacaagaa gaagacattc 240
cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa gaccaccaaa 300
cttcttatcc ctgccgctag tttcatccga gaagtgagaa gtgtcaccca gatctttgcc 360
cctcccgatg ttacccgttg gactgctgaa gcttttatgg ctattcaaga ggcggctgaa 420
gattttttaa ttggcttgtt ctctgatgct atgctttgcg ctatccatgc aaggcgtgtt 480
actctaatga gaaaagattt tgagcttgca cgccgtcttg gaggaaaagg cagaccattg 540
<210> 70
<211> 180
<212> PRT
<213> Brassica napus
<400> 70
Met Ala Arg Thr Lys His Phe Ala Ser Arg Ala Arg Asp Arg Asn Arg
1 5 10 15
Thr Asn Ala Thr Ala Ser Ser Ser Ala Ala Ala Ala Glu Gly Pro Ser
20 25 30
Ala Thr Pro Thr Arg Arg Glu Gly Ser Gln Gly Glu Ala Gln Gln Thr
35 40 45
Thr Pro Thr Thr Thr Pro Pro Ala Gly Arg Lys Lys Gly Gly Thr Lys
50 55 60
Arg Thr Lys Gln Ala Met Pro Lys Ser Ser Asn Lys Lys Lys Thr Phe
65 70 75 80
Arg Tyr Lys Pro Gly Thr Val Ala Leu Arg Glu Ile Arg His Phe Gln
85 90 95
Lys Thr Thr Lys Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val
100 105 110
Arg Ser Val Thr Gln Ile Phe Ala Pro Pro Asp Val Thr Arg Trp Thr
115 120 125
Ala Glu Ala Phe Met Ala Ile Gln Glu Ala Ala Glu Asp Phe Leu Ile
130 135 140
Gly Leu Phe Ser Asp Ala Met Leu Cys Ala Ile His Ala Arg Arg Val
145 150 155 160
Thr Leu Met Arg Lys Asp Phe Glu Leu Ala Arg Arg Leu Gly Gly Lys
165 170 175
Gly Arg Pro Leu
180
<210> 71
<211> 4001
<212> DNA
<213> Brassica napus
<400> 71
tgtccgggag gatccaccgg cggtggtcgt tactccattt taacttgatg tcttgaaagc 60
agaggacatg gtatggtggc ggcagaatct atttttagtt gttaattttt cttttcctct 120
gattcttttt atttttttcg aatgaactaa ctttgggttt attcagaaga attatcatct 180
aaaaactgat tcaataaaca aaataattta catatttcac aatgagccat tagtaaacaa 240
gtcgaaagtg aaaccaaatg ggaagagaac aattttaata aaaatatgtt ctaatttcct 300
actttttatg aattgaactc ccgaagagaa tggccgaaga acggagtaaa agctcaatga 360
ttgtaaaagc tatgtattct cttgctttga ggaaaaagct ttttgtttgc actcataggc 420
ctgatatgtt gtgatggctc tttacatatt gggtcttttg tggtctatta aacggttact 480
gaagaattag tttatcgcat ttaaaaaaaa atatttgaaa accacatatc taaaatctca 540
atatattatt tattgataga tgaaaagaaa aatagtttat aaaataatat taattggtaa 600
atggaagtca aaattttaca tttaatacat gactagttcg attctccggc tgaaacccat 660
ttaccactga gccaagacca cttgattata tatatcaagc tcttgttttt tgcattaaca 720
aaagttgcat ccaaaatttc aaaacaacaa ctaaattaat gtctttctat tttcaaagtt 780
ttatctccaa acctattaat agtttaattt ttttttttaa aattaggaaa ccggttaaaa 840
tatattttaa atacaaaaaa cttaaacaat gaatcattta tttatttatt cttcaaaatt 900
taaatatccg aacccggccc aaaatatccg aacccgaaca taaaatatcc gaacccgact 960
cgaagtgtag aaaatatccg aacgggtttt atacctttat actgaaatac cctatacgaa 1020
cccgaatgtg tatccgaacg ccccctacaa tatatgatca tcatttgtat cttgattgaa 1080
caaaaaaaaa gttaaactat tgatcacaaa attttcaatg tgagactttt accattttta 1140
gtcatttata gtcgttttta aaaattcaaa atataactta taagaaaaaa tctaattttt 1200
tttattatat gcttaatgtg attgtttaat ttcttttaat aatataaaat taaacaaaaa 1260
atgagaggtt aaaaaaattg ttatcaaata tgtattattc ataatcatta attgtcatat 1320
atatgttaat tatattaggt aatttcgtag tttttattta agaaaagaaa aaaatattat 1380
tttgtacact actaattaat ttgatagtta gtttaataaa aaatatatta tattattata 1440
tggaccaact tatttttcta aaaaaaaacc actgttttaa aaaccaaacc aactataaac 1500
cggagatata ccggattgag tggctaaaac actctttgta tatatgtgct gagcaaaccc 1560
tctgagtgag atggcgtgtt aagaagtagg aggaccattc atgcctctta tgagttgtag 1620
tctgtgtgta caaaaaagaa gcgttggtgt gaaagaaagc agaaggattt gaaaatcaaa 1680
aaaattgaag gagaagcggg aaaacaaata atctctccct ccgctttttt ttctccaaat 1740
aatcaatctc tcatttcatt tgttaaccca agtttttgat aattatttca aaggggttta 1800
tttatctttt attcctccgg cggcagtaag tagtaatcaa tggcgagaac caaacatttc 1860
gcttccaggg cacgagatcg caatcgaact agttagtact ctctctctct ctgccttttt 1920
tttgatattt attttctagg ttaaacccta atttggcatc tgaaatttgt agatgcgact 1980
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcgg tacgtcatct attttctttt 2040
cccgttttag gtttttacgc aaatctcgtt actgtttttt tgacgaatcg attgaaatgt 2100
gtagaccccg acgagaagag aaggcagcca aggagaagct caacagagtg agtctttcta 2160
tttcattttc tgagatccat gaatcctttt catctctcgt gtgttgtgac atgaatcaat 2220
tgcagcagca actcctacta cgactccacc agccggtaga aaagtaagtt acatttccat 2280
ttcacaccat tcatttgctt ctttatcaac aaactgctct ctcatctgtt ttttttgttt 2340
tgttttggtt ttgtgaagaa aggagggact aagcgaacta aacaagctat gcctaaaagt 2400
tagtgacaga ttttaaaatc tctattttgg atcatcattc tctcaggaca tgtctatttg 2460
catttgttct tattatgtct gtctgtctgt ctttgtcccc cttgtaggtt ccaacaagaa 2520
gaagacattc cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa 2580
gaccaccaaa cttcttatcc ctgccgctag tttcatccga gaagttagta atgaactttg 2640
ttattcatac attcccgctt acttgttttc aatgactctg caattactga tatagaattt 2700
ggagcaacca ttatggggtg atttctctaa ctacaaatta ctaatactat cccaggtgag 2760
aagtgtcacc cagatctttg cccctcccga tgttacccgt tggactgctg aagctcttat 2820
ggctattcaa gaggtacgtg tactccttcc ctcttttgtt tcctattttc cacttgatgt 2880
ctaatttaaa ctgatcgttt tttttttata tttcttttgg tgtggggcgg ggcagacggc 2940
tgaagatttt ttaattggct tgttctctga tgctatgctt tgcgctatcc atgcaaggcg 3000
tgttactcta agtaagtagt actccccaaa ataaggaaac ccattttata tacaacattg 3060
cctcatccat gtctgcttct cttcatatca gtgagaaaag attttgagct tgcacgccgt 3120
cttggaggaa aaggcagacc attgtgatcg tttcgcaggt tgtataactt tgttcactcc 3180
ttatgtcttg tcatttgtga tctgactgac actttctttt gaaacataac tgcttgattc 3240
aatatctagg ctgtaaaact tatccctcct tgtttactat cttatatgct ttttccttgg 3300
aattgatagt ttccattgag atttcacttg cacgaaacat atctgctttc tcaatatctc 3360
tcagtcttag aaagggctat tgactaaaag aaaagaaaat ttagaggaag atttgtaaag 3420
acatgtgttt agagagggct taattaaaaa cacacgcttc tgctagcctt gctatttgat 3480
tcccaatttc aacttttttc gaggcatatt ataaagtttt taaatgtact tggcacttca 3540
acttttataa tttatataac gattttattc taatagagca tttgtgattt catagtgttg 3600
tcatgaaact caagtaattc acaccgtccg atgttgctat tgtctaataa aatgttgaaa 3660
aaattgtcaa aacagaacaa aaaacaacat agttgtctct atggtataaa actatcacta 3720
agttgtctct atagtataat atttttcgca atcccaaaac taatttttct ttaatcaaat 3780
taaacataaa ctaaaaccat ttttaaaaag tttaatggaa aaagataaaa aaataaggta 3840
atctcgtaat gttttaaaaa ggaaaaaaaa tgtaaaaaca atttaaaaaa aagaacacac 3900
gacacagatc aaaaatatca tgtaatctaa ttgcatttgg tttctaaaat cttccaaaac 3960
tattctttta aaattctcta aggtaaaact tgattccaat a 4001
<210> 72
<211> 540
<212> DNA
<213> Brassica napus
<400> 72
atggcgagaa ccaaacattt cgcttccagg gcacgagatc gcaatcgaac taatgcgact 60
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcga ccccgacgag aagagaaggc 120
agccaaggag aagctcaaca gacaactcct actacgactc caccagccgg tagaaaaaaa 180
ggagggacta agcgaactaa acaagctatg cctaaaagtt ccaacaagaa gaagacattc 240
cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa gaccaccaaa 300
cttcttatcc ctgccgctag tttcatccga gaagtgagaa gtgtcaccca gatctttgcc 360
cctcccgatg ttacccgttg gactgctgaa gctcttatgg ctattcaaga gacggctgaa 420
gattttttaa ttggcttgtt ctctgatgct atgctttgcg ctatccatgc aaggcgtgtt 480
actctaatga gaaaagattt tgagcttgca cgccgtcttg gaggaaaagg cagaccattg 540
<210> 73
<211> 180
<212> PRT
<213> Brassica napus
<400> 73
Met Ala Arg Thr Lys His Phe Ala Ser Arg Ala Arg Asp Arg Asn Arg
1 5 10 15
Thr Asn Ala Thr Ala Ser Ser Ser Ala Ala Ala Ala Glu Gly Pro Ser
20 25 30
Ala Thr Pro Thr Arg Arg Glu Gly Ser Gln Gly Glu Ala Gln Gln Thr
35 40 45
Thr Pro Thr Thr Thr Pro Pro Ala Gly Arg Lys Lys Gly Gly Thr Lys
50 55 60
Arg Thr Lys Gln Ala Met Pro Lys Ser Ser Asn Lys Lys Lys Thr Phe
65 70 75 80
Arg Tyr Lys Pro Gly Thr Val Ala Leu Arg Glu Ile Arg His Phe Gln
85 90 95
Lys Thr Thr Lys Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val
100 105 110
Arg Ser Val Thr Gln Ile Phe Ala Pro Pro Asp Val Thr Arg Trp Thr
115 120 125
Ala Glu Ala Leu Met Ala Ile Gln Glu Thr Ala Glu Asp Phe Leu Ile
130 135 140
Gly Leu Phe Ser Asp Ala Met Leu Cys Ala Ile His Ala Arg Arg Val
145 150 155 160
Thr Leu Met Arg Lys Asp Phe Glu Leu Ala Arg Arg Leu Gly Gly Lys
165 170 175
Gly Arg Pro Leu
180
<210> 74
<211> 4001
<212> DNA
<213> Brassica napus
<400> 74
tgtccgggag gatccaccgg cggtggtcgt tactccattt taacttgatg tcttgaaagc 60
agaggacatg gtatggtggc ggcagaatct atttttagtt gttaattttt cttttcctct 120
gattcttttt atttttttcg aatgaactaa ctttgggttt attcagaaga attatcatct 180
aaaaactgat tcaataaaca aaataattta catatttcac aatgagccat tagtaaacaa 240
gtcgaaagtg aaaccaaatg ggaagagaac aattttaata aaaatatgtt ctaatttcct 300
actttttatg aattgaactc ccgaagagaa tggccgaaga acggagtaaa agctcaatga 360
ttgtaaaagc tatgtattct cttgctttga ggaaaaagct ttttgtttgc actcataggc 420
ctgatatgtt gtgatggctc tttacatatt gggtcttttg tggtctatta aacggttact 480
gaagaattag tttatcgcat ttaaaaaaaa atatttgaaa accacatatc taaaatctca 540
atatattatt tattgataga tgaaaagaaa aatagtttat aaaataatat taattggtaa 600
atggaagtca aaattttaca tttaatacat gactagttcg attctccggc tgaaacccat 660
ttaccactga gccaagacca cttgattata tatatcaagc tcttgttttt tgcattaaca 720
aaagttgcat ccaaaatttc aaaacaacaa ctaaattaat gtctttctat tttcaaagtt 780
ttatctccaa acctattaat agtttaattt ttttttttaa aattaggaaa ccggttaaaa 840
tatattttaa atacaaaaaa cttaaacaat gaatcattta tttatttatt cttcaaaatt 900
taaatatccg aacccggccc aaaatatccg aacccgaaca taaaatatcc gaacccgact 960
cgaagtgtag aaaatatccg aacgggtttt atacctttat actgaaatac cctatacgaa 1020
cccgaatgtg tatccgaacg ccccctacaa tatatgatca tcatttgtat cttgattgaa 1080
caaaaaaaaa gttaaactat tgatcacaaa attttcaatg tgagactttt accattttta 1140
gtcatttata gtcgttttta aaaattcaaa atataactta taagaaaaaa tctaattttt 1200
tttattatat gcttaatgtg attgtttaat ttcttttaat aatataaaat taaacaaaaa 1260
atgagaggtt aaaaaaattg ttatcaaata tgtattattc ataatcatta attgtcatat 1320
atatgttaat tatattaggt aatttcgtag tttttattta agaaaagaaa aaaatattat 1380
tttgtacact actaattaat ttgatagtta gtttaataaa aaatatatta tattattata 1440
tggaccaact tatttttcta aaaaaaaacc actgttttaa aaaccaaacc aactataaac 1500
cggagatata ccggattgag tggctaaaac actctttgta tatatgtgct gagcaaaccc 1560
tctgagtgag atggcgtgtt aagaagtagg aggaccattc atgcctctta tgagttgtag 1620
tctgtgtgta caaaaaagaa gcgttggtgt gaaagaaagc agaaggattt gaaaatcaaa 1680
aaaattgaag gagaagcggg aaaacaaata atctctccct ccgctttttt ttctccaaat 1740
aatcaatctc tcatttcatt tgttaaccca agtttttgat aattatttca aaggggttta 1800
tttatctttt attcctccgg cggcagtaag tagtaatcaa tggcgagaac caaacatttc 1860
gcttccaggg cacgagatcg caatcgaact agttagtact ctctctctct ctgccttttt 1920
tttgatattt attttctagg ttaaacccta atttggcatc tgaaatttgt agatgcgact 1980
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcgg tacgtcatct attttctttt 2040
cccgttttag gtttttacgc aaatctcgtt actgtttttt tgacgaatcg attgaaatgt 2100
gtagaccccg acgagaagag aaggcagcca aggagaagct caacagagtg agtctttcta 2160
tttcattttc tgagatccat gaatcctttt catctctcgt gtgttgtgac atgaatcaat 2220
tgcagcagca actcctacta cgactccacc agccggtaga aaagtaagtt acatttccat 2280
ttcacaccat tcatttgctt ctttatcaac aaactgctct ctcatctgtt ttttttgttt 2340
tgttttggtt ttgtgaagaa aggagggact aagcgaacta aacaagctat gcctaaaagt 2400
tagtgacaga ttttaaaatc tctattttgg atcatcattc tctcaggaca tgtctatttg 2460
catttgttct tattatgtct gtctgtctgt ctttgtcccc cttgtaggtt ccaacaagaa 2520
gaagacattc cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa 2580
gaccaccaaa cttcttatcc ctgccgctag tttcatccga gaagttagta atgaactttg 2640
ttattcatac attcccgctt acttgttttc aatgactctg caattactga tatagaattt 2700
ggagcaacca ttatggggtg atttctctaa ctacaaatta ctaatactat cccaggtgag 2760
aagtgtcacc cagatctttg cccctcccga tgttacccgt tggactgctg aagctcttat 2820
ggctattcaa gaggtacgtg tactccttcc ctcttttgtt tcctattttc cacttgatgt 2880
ctaatttaaa ctgatcgttt tttttttata tttcttttgg tgtggggcgg ggcaggcggc 2940
tgaagatttt ttaattggct tgttctctga tactatgctt tgcgctatcc atgcaaggcg 3000
tgttactcta agtaagtagt actccccaaa ataaggaaac ccattttata tacaacattg 3060
cctcatccat gtctgcttct cttcatatca gtgagaaaag attttgagct tgcacgccgt 3120
cttggaggaa aaggcagacc attgtgatcg tttcgcaggt tgtataactt tgttcactcc 3180
ttatgtcttg tcatttgtga tctgactgac actttctttt gaaacataac tgcttgattc 3240
aatatctagg ctgtaaaact tatccctcct tgtttactat cttatatgct ttttccttgg 3300
aattgatagt ttccattgag atttcacttg cacgaaacat atctgctttc tcaatatctc 3360
tcagtcttag aaagggctat tgactaaaag aaaagaaaat ttagaggaag atttgtaaag 3420
acatgtgttt agagagggct taattaaaaa cacacgcttc tgctagcctt gctatttgat 3480
tcccaatttc aacttttttc gaggcatatt ataaagtttt taaatgtact tggcacttca 3540
acttttataa tttatataac gattttattc taatagagca tttgtgattt catagtgttg 3600
tcatgaaact caagtaattc acaccgtccg atgttgctat tgtctaataa aatgttgaaa 3660
aaattgtcaa aacagaacaa aaaacaacat agttgtctct atggtataaa actatcacta 3720
agttgtctct atagtataat atttttcgca atcccaaaac taatttttct ttaatcaaat 3780
taaacataaa ctaaaaccat ttttaaaaag tttaatggaa aaagataaaa aaataaggta 3840
atctcgtaat gttttaaaaa ggaaaaaaaa tgtaaaaaca atttaaaaaa aagaacacac 3900
gacacagatc aaaaatatca tgtaatctaa ttgcatttgg tttctaaaat cttccaaaac 3960
tattctttta aaattctcta aggtaaaact tgattccaat a 4001
<210> 75
<211> 540
<212> DNA
<213> Brassica napus
<400> 75
atggcgagaa ccaaacattt cgcttccagg gcacgagatc gcaatcgaac taatgcgact 60
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcga ccccgacgag aagagaaggc 120
agccaaggag aagctcaaca gacaactcct actacgactc caccagccgg tagaaaaaaa 180
ggagggacta agcgaactaa acaagctatg cctaaaagtt ccaacaagaa gaagacattc 240
cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa gaccaccaaa 300
cttcttatcc ctgccgctag tttcatccga gaagtgagaa gtgtcaccca gatctttgcc 360
cctcccgatg ttacccgttg gactgctgaa gctcttatgg ctattcaaga ggcggctgaa 420
gattttttaa ttggcttgtt ctctgatact atgctttgcg ctatccatgc aaggcgtgtt 480
actctaatga gaaaagattt tgagcttgca cgccgtcttg gaggaaaagg cagaccattg 540
<210> 76
<211> 180
<212> PRT
<213> Brassica napus
<400> 76
Met Ala Arg Thr Lys His Phe Ala Ser Arg Ala Arg Asp Arg Asn Arg
1 5 10 15
Thr Asn Ala Thr Ala Ser Ser Ser Ala Ala Ala Ala Glu Gly Pro Ser
20 25 30
Ala Thr Pro Thr Arg Arg Glu Gly Ser Gln Gly Glu Ala Gln Gln Thr
35 40 45
Thr Pro Thr Thr Thr Pro Pro Ala Gly Arg Lys Lys Gly Gly Thr Lys
50 55 60
Arg Thr Lys Gln Ala Met Pro Lys Ser Ser Asn Lys Lys Lys Thr Phe
65 70 75 80
Arg Tyr Lys Pro Gly Thr Val Ala Leu Arg Glu Ile Arg His Phe Gln
85 90 95
Lys Thr Thr Lys Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val
100 105 110
Arg Ser Val Thr Gln Ile Phe Ala Pro Pro Asp Val Thr Arg Trp Thr
115 120 125
Ala Glu Ala Leu Met Ala Ile Gln Glu Ala Ala Glu Asp Phe Leu Ile
130 135 140
Gly Leu Phe Ser Asp Ala Met Leu Tyr Ala Ile His Ala Arg Arg Val
145 150 155 160
Thr Leu Met Arg Lys Asp Phe Glu Leu Ala Arg Arg Leu Gly Gly Lys
165 170 175
Gly Arg Pro Leu
180
<210> 77
<211> 4001
<212> DNA
<213> Brassica napus
<400> 77
tgtccgggag gatccaccgg cggtggtcgt tactccattt taacttgatg tcttgaaagc 60
agaggacatg gtatggtggc ggcagaatct atttttagtt gttaattttt cttttcctct 120
gattcttttt atttttttcg aatgaactaa ctttgggttt attcagaaga attatcatct 180
aaaaactgat tcaataaaca aaataattta catatttcac aatgagccat tagtaaacaa 240
gtcgaaagtg aaaccaaatg ggaagagaac aattttaata aaaatatgtt ctaatttcct 300
actttttatg aattgaactc ccgaagagaa tggccgaaga acggagtaaa agctcaatga 360
ttgtaaaagc tatgtattct cttgctttga ggaaaaagct ttttgtttgc actcataggc 420
ctgatatgtt gtgatggctc tttacatatt gggtcttttg tggtctatta aacggttact 480
gaagaattag tttatcgcat ttaaaaaaaa atatttgaaa accacatatc taaaatctca 540
atatattatt tattgataga tgaaaagaaa aatagtttat aaaataatat taattggtaa 600
atggaagtca aaattttaca tttaatacat gactagttcg attctccggc tgaaacccat 660
ttaccactga gccaagacca cttgattata tatatcaagc tcttgttttt tgcattaaca 720
aaagttgcat ccaaaatttc aaaacaacaa ctaaattaat gtctttctat tttcaaagtt 780
ttatctccaa acctattaat agtttaattt ttttttttaa aattaggaaa ccggttaaaa 840
tatattttaa atacaaaaaa cttaaacaat gaatcattta tttatttatt cttcaaaatt 900
taaatatccg aacccggccc aaaatatccg aacccgaaca taaaatatcc gaacccgact 960
cgaagtgtag aaaatatccg aacgggtttt atacctttat actgaaatac cctatacgaa 1020
cccgaatgtg tatccgaacg ccccctacaa tatatgatca tcatttgtat cttgattgaa 1080
caaaaaaaaa gttaaactat tgatcacaaa attttcaatg tgagactttt accattttta 1140
gtcatttata gtcgttttta aaaattcaaa atataactta taagaaaaaa tctaattttt 1200
tttattatat gcttaatgtg attgtttaat ttcttttaat aatataaaat taaacaaaaa 1260
atgagaggtt aaaaaaattg ttatcaaata tgtattattc ataatcatta attgtcatat 1320
atatgttaat tatattaggt aatttcgtag tttttattta agaaaagaaa aaaatattat 1380
tttgtacact actaattaat ttgatagtta gtttaataaa aaatatatta tattattata 1440
tggaccaact tatttttcta aaaaaaaacc actgttttaa aaaccaaacc aactataaac 1500
cggagatata ccggattgag tggctaaaac actctttgta tatatgtgct gagcaaaccc 1560
tctgagtgag atggcgtgtt aagaagtagg aggaccattc atgcctctta tgagttgtag 1620
tctgtgtgta caaaaaagaa gcgttggtgt gaaagaaagc agaaggattt gaaaatcaaa 1680
aaaattgaag gagaagcggg aaaacaaata atctctccct ccgctttttt ttctccaaat 1740
aatcaatctc tcatttcatt tgttaaccca agtttttgat aattatttca aaggggttta 1800
tttatctttt attcctccgg cggcagtaag tagtaatcaa tggcgagaac caaacatttc 1860
gcttccaggg cacgagatcg caatcgaact agttagtact ctctctctct ctgccttttt 1920
tttgatattt attttctagg ttaaacccta atttggcatc tgaaatttgt agatgcgact 1980
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcgg tacgtcatct attttctttt 2040
cccgttttag gtttttacgc aaatctcgtt actgtttttt tgacgaatcg attgaaatgt 2100
gtagaccccg acgagaagag aaggcagcca aggagaagct caacagagtg agtctttcta 2160
tttcattttc tgagatccat gaatcctttt catctctcgt gtgttgtgac atgaatcaat 2220
tgcagcagca actcctacta cgactccacc agccggtaga aaagtaagtt acatttccat 2280
ttcacaccat tcatttgctt ctttatcaac aaactgctct ctcatctgtt ttttttgttt 2340
tgttttggtt ttgtgaagaa aggagggact aagcgaacta aacaagctat gcctaaaagt 2400
tagtgacaga ttttaaaatc tctattttgg atcatcattc tctcaggaca tgtctatttg 2460
catttgttct tattatgtct gtctgtctgt ctttgtcccc cttgtaggtt ccaacaagaa 2520
gaagacattc cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa 2580
gaccaccaaa cttcttatcc ctgccgctag tttcatccga gaagttagta atgaactttg 2640
ttattcatac attcccgctt acttgttttc aatgactctg caattactga tatagaattt 2700
ggagcaacca ttatggggtg atttctctaa ctacaaatta ctaatactat cccaggtgag 2760
aagtgtcacc cagatctttg cccctcccga tgttacccgt tggactgctg aagctcttat 2820
ggctattcaa gaggtacgtg tactccttcc ctcttttgtt tcctattttc cacttgatgt 2880
ctaatttaaa ctgatcgttt tttttttata tttcttttgg tgtggggcgg ggcaggcggc 2940
tgaagatttt ttaattggct tgttctctga tgctatgttt tgcgctatcc atgcaaggcg 3000
tgttactcta agtaagtagt actccccaaa ataaggaaac ccattttata tacaacattg 3060
cctcatccat gtctgcttct cttcatatca gtgagaaaag attttgagct tgcacgccgt 3120
cttggaggaa aaggcagacc attgtgatcg tttcgcaggt tgtataactt tgttcactcc 3180
ttatgtcttg tcatttgtga tctgactgac actttctttt gaaacataac tgcttgattc 3240
aatatctagg ctgtaaaact tatccctcct tgtttactat cttatatgct ttttccttgg 3300
aattgatagt ttccattgag atttcacttg cacgaaacat atctgctttc tcaatatctc 3360
tcagtcttag aaagggctat tgactaaaag aaaagaaaat ttagaggaag atttgtaaag 3420
acatgtgttt agagagggct taattaaaaa cacacgcttc tgctagcctt gctatttgat 3480
tcccaatttc aacttttttc gaggcatatt ataaagtttt taaatgtact tggcacttca 3540
acttttataa tttatataac gattttattc taatagagca tttgtgattt catagtgttg 3600
tcatgaaact caagtaattc acaccgtccg atgttgctat tgtctaataa aatgttgaaa 3660
aaattgtcaa aacagaacaa aaaacaacat agttgtctct atggtataaa actatcacta 3720
agttgtctct atagtataat atttttcgca atcccaaaac taatttttct ttaatcaaat 3780
taaacataaa ctaaaaccat ttttaaaaag tttaatggaa aaagataaaa aaataaggta 3840
atctcgtaat gttttaaaaa ggaaaaaaaa tgtaaaaaca atttaaaaaa aagaacacac 3900
gacacagatc aaaaatatca tgtaatctaa ttgcatttgg tttctaaaat cttccaaaac 3960
tattctttta aaattctcta aggtaaaact tgattccaat a 4001
<210> 78
<211> 540
<212> DNA
<213> Brassica napus
<400> 78
atggcgagaa ccaaacattt cgcttccagg gcacgagatc gcaatcgaac taatgcgact 60
gcttcatctt cggcggcggc ggcggaaggt ccgagtgcga ccccgacgag aagagaaggc 120
agccaaggag aagctcaaca gacaactcct actacgactc caccagccgg tagaaaaaaa 180
ggagggacta agcgaactaa acaagctatg cctaaaagtt ccaacaagaa gaagacattc 240
cgttacaagc ctggaaccgt tgccctcaga gagattcgcc atttccagaa gaccaccaaa 300
cttcttatcc ctgccgctag tttcatccga gaagtgagaa gtgtcaccca gatctttgcc 360
cctcccgatg ttacccgttg gactgctgaa gctcttatgg ctattcaaga ggcggctgaa 420
gattttttaa ttggcttgtt ctctgatgct atgttttgcg ctatccatgc aaggcgtgtt 480
actctaatga gaaaagattt tgagcttgca cgccgtcttg gaggaaaagg cagaccattg 540
<210> 79
<211> 180
<212> PRT
<213> Brassica napus
<400> 79
Met Ala Arg Thr Lys His Phe Ala Ser Arg Ala Arg Asp Arg Asn Arg
1 5 10 15
Thr Asn Ala Thr Ala Ser Ser Ser Ala Ala Ala Ala Glu Gly Pro Ser
20 25 30
Ala Thr Pro Thr Arg Arg Glu Gly Ser Gln Gly Glu Ala Gln Gln Thr
35 40 45
Thr Pro Thr Thr Thr Pro Pro Ala Gly Arg Lys Lys Gly Gly Thr Lys
50 55 60
Arg Thr Lys Gln Ala Met Pro Lys Ser Ser Asn Lys Lys Lys Thr Phe
65 70 75 80
Arg Tyr Lys Pro Gly Thr Val Ala Leu Arg Glu Ile Arg His Phe Gln
85 90 95
Lys Thr Thr Lys Leu Leu Ile Pro Ala Ala Ser Phe Ile Arg Glu Val
100 105 110
Arg Ser Val Thr Gln Ile Phe Ala Pro Pro Asp Val Thr Arg Trp Thr
115 120 125
Ala Glu Ala Leu Met Ala Ile Gln Glu Ala Ala Glu Asp Phe Leu Ile
130 135 140
Gly Leu Phe Ser Asp Ala Met Leu Cys Val Ile His Ala Arg Arg Val
145 150 155 160
Thr Leu Met Arg Lys Asp Phe Glu Leu Ala Arg Arg Leu Gly Gly Lys
165 170 175
Gly Arg Pro Leu
180
<210> 80
<211> 5834
<212> DNA
<213> Zea mays
<400> 80
ttatgtagag gcaattgcag tagtgcctct gttttagagt gtaactacag atttgtccct 60
atttttttag agtttgcgtg tttgtccctg ttttttcaaa tcaaactatt gtatacccct 120
actccattag ttatacttaa caatgttaag tcttgataaa aagacaaggg ataattggat 180
tagtgaccct gttttagagt gtaattatag ctttgcccga tgttttagac ttcacatgtt 240
tttatgacaa ttcaaattgt ttccataaca tcttaaatta ttttgacaac atttagaatt 300
gttttgcaat aatttaaatt atttccaaaa taaaaatatt ttgacaatta ttttatcaac 360
aaattaaatt atttttttta caaaataatt tgtcaaggta cttttttaaa attttgaaaa 420
taatcaaatt attgtaaata taatttgaat tgttgcgaaa ataatttgga ttgtcataaa 480
aacacgtaag tctaaaaatt aggcgtaaaa ctacaattat actctaaaaa ggtggtaatg 540
gcgtagttgt tccttgtcta ttttatcaag acatagcacc gtgcagtaca actaatggag 600
tagtgacata caacaatttt gttttaaata ataggggtaa atacgcaaag tctaaaaaac 660
agaggtaaat ctacaattac atgttaaaat agaggcatcg atacaattgt accttttata 720
tagcagcatg cgccctgttg ggatacaatt gtacctttca catgtcttct agatggttcc 780
caaccctttg gccaagatcg tacagataat attgcgagga gcccaaatca acggtgtcca 840
tatgttatgt tgatgtggat ggtttaccta ggcgcaaaag tgcgctggtt tcgtccgtac 900
aaatatactt taagtatggt tttgattttt ttctattttt cattttttaa ataaaacgag 960
acaatcaaat ctgatataaa aatcaaatga attataaata gagacggaaa gagtatatat 1020
atttgttttg ctattattta aagtattaaa agatagtgga cgaatgaacg tcctctatgt 1080
ttaaaagaac gttttagagg acgttgtgtt gttgaaggaa atatgaaaaa aaaatcttct 1140
gcatatttag aagggaggag cgtttacaca ttactttcgg gacttcaacc caaatatgtc 1200
aaggtttgtg agtggctcag tgcggaaaaa aaatcctata tataccagat gtaaacacta 1260
tcttttacag cctatcacat tcacatttag aggttcacaa agatagatca aaatttataa 1320
aataatcatt taatattttt tttattttat ttatatggat aagcagctgg tgtatgtgag 1380
gagctgtaaa agatattttt tacatccgag atgtaaagat tttttttaac tcaatgctgg 1440
ttaccggctg ggaggacgat gataaagaaa gcatctctca ctgcattccg ggcccactac 1500
tcaaacgttc ggcacgccag gttggcaggt agccgttaca tcgataggca ctcggccact 1560
cgcacgcaga caccacacca gtgtgctcag tgctcactgc tcaccataat aacgctgcac 1620
ctcttttcat ttcaccatct cctgccccct taaaaaaaag actcaccgtc gacacgccct 1680
cccgtcccga gagttctgaa tcgaaaccgt cggccacgag agcagtgcga ggcgcccacc 1740
gcgatggctc gaaccaagca ccaggccgtg aggaagacgg cggagaagcc caagaagaag 1800
ctccagttcg agcgctcagg taacccgggt cccgcgctcc cccccgcttc gcaagcagac 1860
gctgtcgctt ctctccgacc ctggtgctaa gcacgttcct tgttccgtct tttgcaggtg 1920
gtgcgagtac ctcggcgacg ccggtgagcg cgtgcgtgcg gggatcagtt ccctcctttt 1980
gccttttttt gttgggctgc tcttacttgc ttgcaagctg tttgatggaa tgcaggaaag 2040
ggctgctggg accgggggaa gagcggcgtc tggaggtgac tcaggtgagg acctatttgt 2100
cgttgctgga tgctgggttt cgcttgcaat ctaattttgt tgcaagatga gggcgaatgt 2160
gccagttcca tgtgggtgtc atggtctcgg agttactacc ttaattgctc accatagtat 2220
gttttcttaa aaaaaacagt taagaagacg aaaccacgcc accgctggcg gccagggact 2280
gtagcgctgc gggagatcag gaagtaccag aagtccactg aaccgctcat cccctttgcg 2340
cctttcgtcc gtgtggtggg tgcaggcgtg tttgtcctct gcatagtatg gggttgttcc 2400
gcattctgtc taatggaaag ttattcttct gagaaaaaaa atgcaggtga gggagttaac 2460
caatttcgta acaaacggga aagtagagcg ctataccaca gaagccctcc ttgcgctgca 2520
agaggtcagt tatgaaaaat gtcttatctc tctgttaaga tcctcttcat atacatagtt 2580
gctattgcta tcgtgaagtc ttttttttct gttaattggt ctggtactac ttactagtca 2640
ggatttcata ttgcggtttt tcctagtggt gtgtagttaa aaagtagttt aattgctttt 2700
agttaaaagg ggtgttcagg gctaaagatc aactatgaga aaacagaaat tttcccaatt 2760
cgatacccga cagcattatg gcctgcgcta atggaggtgt ttccgggcaa atactctagc 2820
ctacctggga agtaccttgg gttgcccctt catttcagga aagtaaaaag gaatgatctt 2880
caacctctaa tcgaaaaaat caacaacagg ctggccttgc tggaaaggca agatgttgtc 2940
caaggctggt atagaaactc ttgtaaaatc gatgctatcc gcacaaccaa tctaccatct 3000
aatggttttt ccacctcaaa aatggctgct gcaaacaatt gacaaaatac gaagaaactt 3060
cctgtggaga gggagcaatc cagaagtttg cagcgggggt cactgcctcg tcaactggcc 3120
cgtaacttgc ctcccaaaga acaagggagg tcttggaatt ctggaccttg atcgttttgc 3180
gagggggcta agactaagat ggctgtggct acgatggaag agcaaagata gggcgtggac 3240
tgccttgaag cttccttgtg acaaaactga tgaagatctc ttcaatgctt ccacaactgt 3300
cacggtaggc aatggaaaga tagctgaatt ctggaattct agttggatcc aaggccaagc 3360
ccctaagaac attgcgccaa cactgttcaa gaaggaaaag aggaagaaca tcacggtcgc 3420
caaagcgctc actaacaaca attggattcg tttatgctca ccatacacgg gtgaggggga 3480
gtttagagag gtcgtctctc tttggcaggc cataggtaac atgcaagagc ttaacggttt 3540
ggaagacaac atctcttgga gatggacggc agatgggcag tacagtgcta gcagtgcata 3600
caaaatccag ttcgcatcca atttcactaa aatgaacctc tgccctattt ggaaggctaa 3660
agtggaaccg aaatgccgat tttttgcttg gacactactt cataagagaa ttctgactgc 3720
cgataacctt cataaaagag gttgcaactc agcctcagaa acaattcccc acttatgcaa 3780
ggattgcccc tttagtagag aggtgtggaa caaagttttg tctcgggcca actttccttt 3840
actgactggg tctcccagtg acacttcttt gtatgattgg tggacggaca tgtgcagcct 3900
ttgcagcaga caggcaagaa gaggtttcga cggtctgcta tttcactttt ggtggaactt 3960
atggctggaa agaaataaca gaatctttca aaggcagcgt agaagtgtag atcaagttgc 4020
tctggcagtc aaggattatg ctagtagctg aagtctagtt ggtttggact agtggttttg 4080
ttgcttttct ttttaatttc tttttagttc tttttatgtt gttttcgttt ccttaagttg 4140
cttggagtct gtattatcct ctttcttcta atatagatcg gagcgacaaa ccttttgccc 4200
cttcctttca aaaaaaagtt aaaagggaat ttaactgctt tcctagtggt gtagttaaaa 4260
tggatttcat attgcggcct ttcctagctt gcttgctatt gattggacta tagtgatcca 4320
aatgctgata actttgtcgc ttgtgtaggc atggttagag agcttagagt ttgcatttat 4380
tcaatacctt gagactgcat ttcatataca taaattattc atgattattt cttttctcta 4440
tttgttctgg ttaattaaga gttttaggtt tccatatttt tgtacgtgca tcatttaaat 4500
tcttgtattg tttttcgttc ttgtctacag gcagcagaat tccacttgat agaactgttt 4560
gaaatggcga atctgtgtgc catccatgcc aagcgtgtca caatcagtaa gttatcactg 4620
agtgaactcc tttttctctg tagcattact cctaatgaat atgtgtgatg cattttggtt 4680
gcacgattct ttagtgattc tgcttcagat ggatatgata aatctagatg ttattttgaa 4740
gtggcgaatt gcttacgagc ggaaatagta atgttcaaat agcgcaaagt gcaactgttg 4800
acttttagta ggccatttat atggtttgat taccaacaaa tacgtcaatc atatgatttg 4860
attatcaaca aaggaatcag ctatatggtt tgattatcaa caaaggaatc agctaggttt 4920
gcttatcaac attcaacaaa ggcatcaagt aatactccat ccgtttcaat ttataattcg 4980
tttgactttt tttatctaag tttgatcggc tcgacttatt aaaaaaaatc ataattattg 5040
ttaatttttg ttgtgatatt gtttagtata atatacttta aatgtgactt tgagtttttc 5100
attttttcgc aaaaaaaaat gaataggacg agccggtcaa acgtgacaca aaaaagtcaa 5160
acgaattata atttgggaca cacggagtag taaataatgt aacaacttag agagtgggac 5220
aaaaaaatct ctagtggtgc taaatttagt tcagctttgt ataaacacaa gcattgattg 5280
agaaatctga caactcaagg atctgtagga aatgtgttac cctaaatgtt ttccttactg 5340
atgcagtgca aaaggacata caacttgcaa ggcgtatcgg aggaaggcgt tgggcatgat 5400
atataatatc cattctgatt gcatcattct tgtgaatttg tttgtaggag ctagacatta 5460
gtgttgttga atgctgcatg gttcctaatc cttttcgcag tctaacatct gtggagttag 5520
tatgttacat ggcaacagct gaacatctgt ggactatatg gcaacagccg aagattgtgt 5580
ctgtgggata actggttgtt ttggttgctc ttcagtagtt tgtttgcttc aggtaaccat 5640
gctgcgaact atgatgtttt cattctcggt ttgcttcagc taaccgagat cgattcagtc 5700
tgcagtatgg actatggagt aaactgcatg ctgaaacccg aaccactgct gaaactgcat 5760
gctgaaaccc gaaccactgc tacggcagtt gccaggatag caggagggcc tttatgcaca 5820
gtggaattga gtag 5834
<210> 81
<211> 471
<212> DNA
<213> Zea mays
<400> 81
atggctcgaa ccaagcacca ggccgtgagg aagacggcgg agaagcccaa gaagaagctc 60
cagttcgagc gctcaggtgg tgcgagtacc tcggcgacgc cggaaagggc tgctgggacc 120
gggggaagag cggcgtctgg aggtgactca gttaagaaga cgaaaccacg ccaccgctgg 180
cggccaggga ctgtagcgct gcgggagatc aggaagtacc agaagtccac tgaaccgctc 240
atcccctttg cgcctttcgt ccgtgtggtg agggagttaa ccaatttcgt aacaaacggg 300
aaagtagagc gctataccac agaagccctc cttgcgctgc aagaggcagc agaattccac 360
ttgatagaac tgtttgaaat ggcgaatctg tgtgccatcc atgccaagcg tgtcacaatc 420
atgcaaaagg acatacaact tgcaaggcgt atcggaggaa ggcgttgggc a 471
<210> 82
<211> 157
<212> PRT
<213> Zea mays
<400> 82
Met Ala Arg Thr Lys His Gln Ala Val Arg Lys Thr Ala Glu Lys Pro
1 5 10 15
Lys Lys Lys Leu Gln Phe Glu Arg Ser Gly Gly Ala Ser Thr Ser Ala
20 25 30
Thr Pro Glu Arg Ala Ala Gly Thr Gly Gly Arg Ala Ala Ser Gly Gly
35 40 45
Asp Ser Val Lys Lys Thr Lys Pro Arg His Arg Trp Arg Pro Gly Thr
50 55 60
Val Ala Leu Arg Glu Ile Arg Lys Tyr Gln Lys Ser Thr Glu Pro Leu
65 70 75 80
Ile Pro Phe Ala Pro Phe Val Arg Val Val Arg Glu Leu Thr Asn Phe
85 90 95
Val Thr Asn Gly Lys Val Glu Arg Tyr Thr Thr Glu Ala Leu Leu Ala
100 105 110
Leu Gln Glu Ala Ala Glu Phe His Leu Ile Glu Leu Phe Glu Met Ala
115 120 125
Asn Leu Cys Ala Ile His Ala Lys Arg Val Thr Ile Met Gln Lys Asp
130 135 140
Ile Gln Leu Ala Arg Arg Ile Gly Gly Arg Arg Trp Ala
145 150 155
<210> 83
<211> 5834
<212> DNA
<213> Zea mays
<400> 83
ttatgtagag gcaattgcag tagtgcctct gttttagagt gtaactacag atttgtccct 60
atttttttag agtttgcgtg tttgtccctg ttttttcaaa tcaaactatt gtatacccct 120
actccattag ttatacttaa caatgttaag tcttgataaa aagacaaggg ataattggat 180
tagtgaccct gttttagagt gtaattatag ctttgcccga tgttttagac ttcacatgtt 240
tttatgacaa ttcaaattgt ttccataaca tcttaaatta ttttgacaac atttagaatt 300
gttttgcaat aatttaaatt atttccaaaa taaaaatatt ttgacaatta ttttatcaac 360
aaattaaatt atttttttta caaaataatt tgtcaaggta cttttttaaa attttgaaaa 420
taatcaaatt attgtaaata taatttgaat tgttgcgaaa ataatttgga ttgtcataaa 480
aacacgtaag tctaaaaatt aggcgtaaaa ctacaattat actctaaaaa ggtggtaatg 540
gcgtagttgt tccttgtcta ttttatcaag acatagcacc gtgcagtaca actaatggag 600
tagtgacata caacaatttt gttttaaata ataggggtaa atacgcaaag tctaaaaaac 660
agaggtaaat ctacaattac atgttaaaat agaggcatcg atacaattgt accttttata 720
tagcagcatg cgccctgttg ggatacaatt gtacctttca catgtcttct agatggttcc 780
caaccctttg gccaagatcg tacagataat attgcgagga gcccaaatca acggtgtcca 840
tatgttatgt tgatgtggat ggtttaccta ggcgcaaaag tgcgctggtt tcgtccgtac 900
aaatatactt taagtatggt tttgattttt ttctattttt cattttttaa ataaaacgag 960
acaatcaaat ctgatataaa aatcaaatga attataaata gagacggaaa gagtatatat 1020
atttgttttg ctattattta aagtattaaa agatagtgga cgaatgaacg tcctctatgt 1080
ttaaaagaac gttttagagg acgttgtgtt gttgaaggaa atatgaaaaa aaaatcttct 1140
gcatatttag aagggaggag cgtttacaca ttactttcgg gacttcaacc caaatatgtc 1200
aaggtttgtg agtggctcag tgcggaaaaa aaatcctata tataccagat gtaaacacta 1260
tcttttacag cctatcacat tcacatttag aggttcacaa agatagatca aaatttataa 1320
aataatcatt taatattttt tttattttat ttatatggat aagcagctgg tgtatgtgag 1380
gagctgtaaa agatattttt tacatccgag atgtaaagat tttttttaac tcaatgctgg 1440
ttaccggctg ggaggacgat gataaagaaa gcatctctca ctgcattccg ggcccactac 1500
tcaaacgttc ggcacgccag gttggcaggt agccgttaca tcgataggca ctcggccact 1560
cgcacgcaga caccacacca gtgtgctcag tgctcactgc tcaccataat aacgctgcac 1620
ctcttttcat ttcaccatct cctgccccct taaaaaaaag actcaccgtc gacacgccct 1680
cccgtcccga gagttctgaa tcgaaaccgt cggccacgag agcagtgcga ggcgcccacc 1740
gcgatggctc gaaccaagca ccaggccgtg aggaagacgg cggagaagcc caagaagaag 1800
ctccagttcg agcgctcagg taacccgggt cccgcgctcc cccccgcttc gcaagcagac 1860
gctgtcgctt ctctccgacc ctggtgctaa gcacgttcct tgttccgtct tttgcaggtg 1920
gtgcgagtac ctcggcgacg ccggtgagcg cgtgcgtgcg gggatcagtt ccctcctttt 1980
gccttttttt gttgggctgc tcttacttgc ttgcaagctg tttgatggaa tgcaggaaag 2040
ggctgctggg accgggggaa gagcggcgtc tggaggtgac tcaggtgagg acctatttgt 2100
cgttgctgga tgctgggttt cgcttgcaat ctaattttgt tgcaagatga gggcgaatgt 2160
gccagttcca tgtgggtgtc atggtctcgg agttactacc ttaattgctc accatagtat 2220
gttttcttaa aaaaaacagt taagaagacg aaaccacgcc accgctggcg gccagggact 2280
gtagcgctgc gggagatcag gaagtaccag aagtccactg aaccgctcat cccctttgcg 2340
cctttcgtcc gtgtggtggg tgcaggcgtg tttgtcctct gcatagtatg gggttgttcc 2400
gcattctgtc taatggaaag ttattcttct gagaaaaaaa atgcaggtga gggagttaac 2460
caatttcgta acaaacggga aagtagagcg ctataccgca gaagccctcc ttgcgctgta 2520
agaggtcagt tatgaaaaat gtcttatctc tctgttaaga tcctcttcat atacatagtt 2580
gctattgcta tcgtgaagtc ttttttttct gttaattggt ctggtactac ttactagtca 2640
ggatttcata ttgcggtttt tcctagtggt gtgtagttaa aaagtagttt aattgctttt 2700
agttaaaagg ggtgttcagg gctaaagatc aactatgaga aaacagaaat tttcccaatt 2760
cgatacccga cagcattatg gcctgcgcta atggaggtgt ttccgggcaa atactctagc 2820
ctacctggga agtaccttgg gttgcccctt catttcagga aagtaaaaag gaatgatctt 2880
caacctctaa tcgaaaaaat caacaacagg ctggccttgc tggaaaggca agatgttgtc 2940
caaggctggt atagaaactc ttgtaaaatc gatgctatcc gcacaaccaa tctaccatct 3000
aatggttttt ccacctcaaa aatggctgct gcaaacaatt gacaaaatac gaagaaactt 3060
cctgtggaga gggagcaatc cagaagtttg cagcgggggt cactgcctcg tcaactggcc 3120
cgtaacttgc ctcccaaaga acaagggagg tcttggaatt ctggaccttg atcgttttgc 3180
gagggggcta agactaagat ggctgtggct acgatggaag agcaaagata gggcgtggac 3240
tgccttgaag cttccttgtg acaaaactga tgaagatctc ttcaatgctt ccacaactgt 3300
cacggtaggc aatggaaaga tagctgaatt ctggaattct agttggatcc aaggccaagc 3360
ccctaagaac attgcgccaa cactgttcaa gaaggaaaag aggaagaaca tcacggtcgc 3420
caaagcgctc actaacaaca attggattcg tttatgctca ccatacacgg gtgaggggga 3480
gtttagagag gtcgtctctc tttggcaggc cataggtaac atgcaagagc ttaacggttt 3540
ggaagacaac atctcttgga gatggacggc agatgggcag tacagtgcta gcagtgcata 3600
caaaatccag ttcgcatcca atttcactaa aatgaacctc tgccctattt ggaaggctaa 3660
agtggaaccg aaatgccgat tttttgcttg gacactactt cataagagaa ttctgactgc 3720
cgataacctt cataaaagag gttgcaactc agcctcagaa acaattcccc acttatgcaa 3780
ggattgcccc tttagtagag aggtgtggaa caaagttttg tctcgggcca actttccttt 3840
actgactggg tctcccagtg acacttcttt gtatgattgg tggacggaca tgtgcagcct 3900
ttgcagcaga caggcaagaa gaggtttcga cggtctgcta tttcactttt ggtggaactt 3960
atggctggaa agaaataaca gaatctttca aaggcagcgt agaagtgtag atcaagttgc 4020
tctggcagtc aaggattatg ctagtagctg aagtctagtt ggtttggact agtggttttg 4080
ttgcttttct ttttaatttc tttttagttc tttttatgtt gttttcgttt ccttaagttg 4140
cttggagtct gtattatcct ctttcttcta atatagatcg gagcgacaaa ccttttgccc 4200
cttcctttca aaaaaaagtt aaaagggaat ttaactgctt tcctagtggt gtagttaaaa 4260
tggatttcat attgcggcct ttcctagctt gcttgctatt gattggacta tagtgatcca 4320
aatgctgata actttgtcgc ttgtgtaggc atggttagag agcttagagt ttgcatttat 4380
tcaatacctt gagactgcat ttcatataca taaattattc atgattattt cttttctcta 4440
tttgttctgg ttaattaaga gttttaggtt tccatatttt tgtacgtgca tcatttaaat 4500
tcttgtattg tttttcgttc ttgtctacag gcagcagaat tccacttgat agaactgttt 4560
gaaatggcga atctgtgtgc catccatgcc aagcgtgtca caatcagtaa gttatcactg 4620
agtgaactcc tttttctctg tagcattact cctaatgaat atgtgtgatg cattttggtt 4680
gcacgattct ttagtgattc tgcttcagat ggatatgata aatctagatg ttattttgaa 4740
gtggcgaatt gcttacgagc ggaaatagta atgttcaaat agcgcaaagt gcaactgttg 4800
acttttagta ggccatttat atggtttgat taccaacaaa tacgtcaatc atatgatttg 4860
attatcaaca aaggaatcag ctatatggtt tgattatcaa caaaggaatc agctaggttt 4920
gcttatcaac attcaacaaa ggcatcaagt aatactccat ccgtttcaat ttataattcg 4980
tttgactttt tttatctaag tttgatcggc tcgacttatt aaaaaaaatc ataattattg 5040
ttaatttttg ttgtgatatt gtttagtata atatacttta aatgtgactt tgagtttttc 5100
attttttcgc aaaaaaaaat gaataggacg agccggtcaa acgtgacaca aaaaagtcaa 5160
acgaattata atttgggaca cacggagtag taaataatgt aacaacttag agagtgggac 5220
aaaaaaatct ctagtggtgc taaatttagt tcagctttgt ataaacacaa gcattgattg 5280
agaaatctga caactcaagg atctgtagga aatgtgttac cctaaatgtt ttccttactg 5340
atgcagtgca aaaggacata caacttgcaa ggcgtatcgg aggaaggcgt tgggcatgat 5400
atataatatc cattctgatt gcatcattct tgtgaatttg tttgtaggag ctagacatta 5460
gtgttgttga atgctgcatg gttcctaatc cttttcgcag tctaacatct gtggagttag 5520
tatgttacat ggcaacagct gaacatctgt ggactatatg gcaacagccg aagattgtgt 5580
ctgtgggata actggttgtt ttggttgctc ttcagtagtt tgtttgcttc aggtaaccat 5640
gctgcgaact atgatgtttt cattctcggt ttgcttcagc taaccgagat cgattcagtc 5700
tgcagtatgg actatggagt aaactgcatg ctgaaacccg aaccactgct gaaactgcat 5760
gctgaaaccc gaaccactgc tacggcagtt gccaggatag caggagggcc tttatgcaca 5820
gtggaattga gtag 5834
<210> 84
<211> 471
<212> DNA
<213> Zea mays
<400> 84
atggctcgaa ccaagcacca ggccgtgagg aagacggcgg agaagcccaa gaagaagctc 60
cagttcgagc gctcaggtgg tgcgagtacc tcggcgacgc cggaaagggc tgctgggacc 120
gggggaagag cggcgtctgg aggtgactca gttaagaaga cgaaaccacg ccaccgctgg 180
cggccaggga ctgtagcgct gcgggagatc aggaagtacc agaagtccac tgaaccgctc 240
atcccctttg cgcctttcgt ccgtgtggtg agggagttaa ccaatttcgt aacaaacggg 300
aaagtagagc gctataccgc agaagccctc cttgcgctgt aagaggcagc agaattccac 360
ttgatagaac tgtttgaaat ggcgaatctg tgtgccatcc atgccaagcg tgtcacaatc 420
atgcaaaagg acatacaact tgcaaggcgt atcggaggaa ggcgttgggc a 471
<210> 85
<211> 113
<212> PRT
<213> Zea mays
<400> 85
Met Ala Arg Thr Lys His Gln Ala Val Arg Lys Thr Ala Glu Lys Pro
1 5 10 15
Lys Lys Lys Leu Gln Phe Glu Arg Ser Gly Gly Ala Ser Thr Ser Ala
20 25 30
Thr Pro Glu Arg Ala Ala Gly Thr Gly Gly Arg Ala Ala Ser Gly Gly
35 40 45
Asp Ser Val Lys Lys Thr Lys Pro Arg His Arg Trp Arg Pro Gly Thr
50 55 60
Val Ala Leu Arg Glu Ile Arg Lys Tyr Gln Lys Ser Thr Glu Pro Leu
65 70 75 80
Ile Pro Phe Ala Pro Phe Val Arg Val Val Arg Glu Leu Thr Asn Phe
85 90 95
Val Thr Asn Gly Lys Val Glu Arg Tyr Thr Ala Glu Ala Leu Leu Ala
100 105 110
Leu
<210> 86
<211> 3060
<212> DNA
<213> Sorghum bicolor
<400> 86
catctctcac tgccatccgg gtccactact cccaacgttc ggcacgccag gtatagccgt 60
taccccggta ggccccactg gtacacggac aaaggttagc ggtcaccgcg aatcgtgaat 120
acttgtgact acggggtgct aattataaaa acgccgcaca tcctttcgtt tcgccatttc 180
accccccttc ccttcccgta gagaggaaaa aaacccaccg tcgacccgcc cggccgcccg 240
agagttctga atcgaaaccg tcggccgcga ccgcgagagc agcgcggggc gcccaccgtg 300
atggctcgaa ccaagcacca ggccgtgagg aagctgccgc agaagcccaa gaagaagctc 360
cagttcgagc gcgcaggtaa gcccgcgtcc ccgcgctgaa cccccctccg cctcgcgagc 420
agacgctgcc gctgctctcc gtcgcccctg gtgctaagcg cgttcctttt ttttccttct 480
tttgcaggtg gggcgagtac gtcggcgacc ccggtgagtg cgtgcgtgcg tgcgggaatt 540
ggttttagcc ctccttttgc ggtttcgcct tttgttgggc tggtctcact tgcttgcaat 600
ctgtttgatg gaatgcagga gaggaggaat gctgggaccg ggggaggagc cgcggctcgc 660
ggtgaggatc tctttgtcgt tgctgggttt gggaatttcc ggcgcgaaat tatgtggatt 720
tctaggttta tctgccgtct ttcttcttgt cttctctttt ggctctgggg tgagaagtta 780
gggtggttgg gcggacatgg tgcgttattt cgccgtatcg tttggtttgg tgctttctca 840
tccttttaat tccaacatgc cttgtaaaaa ttgcacaaga tttgtttttt catgcatgtc 900
tcagtgttgc taatttgctt ttccggttcg gttggtagaa ttcaatttct tggcgcaata 960
tgcatcttct tttgttgcaa catgagggcg aatgtgccag ttccatatgg gcgtcgcggt 1020
tttgaagtta ctaccttgct tgctcttcgt attataggcg tcattcacaa tagtatgttt 1080
tcttggagat gcagttgcac gggggcgtgt ggagaagaag catcgctggc gggcagggac 1140
tgtagcgctg cgggagatca ggaagtacca gaagtccact gagccgctca tcccctttgc 1200
gcccttcgta cgtgtggtgg gtgcatcttg taccaattgt tgtccactcc atagaatggg 1260
tttgttctgc agtctgtctg atggaaagtt attcttctga gaaaaaatgc aggtcaaaga 1320
gttaactgta ttcataacag actggaggat agggcgctac acccctgaag ccctccttgc 1380
gctgcaagag gtcagttatg aaacatgtct tgtgtatcag ttaagatcat cttctataga 1440
cataattgtt atcatgaagt ctttttctgt taatcggtct ggtactactt aataatcagg 1500
atttcagatt gctgcctttc ctagtggtgt agtcaaaagg gaatttaagt gctgttaggt 1560
actgtttgtt ttggtgtttt gaaccctgcc gcgatcggtt gttgttattc catgtttgtt 1620
tctgtggcag cggacgttca cggtgagatg ggatacgggc gtgtgaaaca tagttacggt 1680
ccatcttcat ggcttatcca tttacgctgc tcgtccgctc acttgttatg tgcggcaacc 1740
aaacttttgt tactagtgta actggtagcg ttgcaaatct ttccatttgc gttaccactc 1800
cctatgggag ccaaacagca ccttagtgta gattccattt gtattacttg agctagcttc 1860
cttgctattg gtgcctcgat tgtactgtta tgatcgaagt gctgaaaact ttgtcgcctg 1920
catagcatga ttagagaact tgagtttaca tttattcaat accttaagac tgcatttcgt 1980
atagataaat tatttttcct aattgttctg gttaactgtt ttaggtttcc atatttttgt 2040
atgtgtatca tttaaattat tgtgttgttt ttcctccctg tctacaggca gcagaattcc 2100
acttgataga actgtttgaa gtggcgaatc tgtgtgccat ccatgccaag cgcgtaacag 2160
tcagtaagtt atcactgaat gaactccttt tcctctgtac tattacgcct aatggagatg 2220
tgtgatgcat ttttggttac acgattcttt agtgattctg cttcagttgg atatgataaa 2280
tctagatgtt atttaaagtg gcaaattgct tacgagtgga aatagtaatg ttcaaatagt 2340
gaaaagtgca attaaacttt taataggcca ttatatggtt tgattgtcaa caaatgcatc 2400
aagaaatagt aaatattata acagttatgg cttagagagt ggacaaaaaa tcggtaatgg 2460
tgagctttgt ataaacacta aaactggctg agaaatctga taactcaagg atctatagga 2520
aatgtattat cctaaatgtt ttccttcctg ctgcagtgca aaaggacata caacttgcaa 2580
ggcgtatcgg aggaaggcgt tggtcgtgat atccattctg attctgatta ccttgttcgg 2640
gtggaatttg tttagaggag ttagacatta gtcttgttga atgctgtgca tggttcctaa 2700
tctgtttcac agttagtggg ctcttctggg atgatctgtt aacacctgtg gagtatgtta 2760
tgtaggaaac acctgaactg aacaacccaa agttgttttg gttgctcttc aaccatttgt 2820
ttgcttcaga gatcgattct aaactgcatg ctaattagtc tatggttgaa caaaaattat 2880
caaatataaa tgaaagtgat atagtagcaa aatccaaaaa aaaaaggatc caaacaaggc 2940
ctaaaatcat ggttctttct ccttttgaac tgggtgcaag tatggacagg cacagaagaa 3000
aaccgcctag caaaccgttt gttttttttt cttcgttgta ccacacgaca ctgttcgttc 3060
<210> 87
<211> 471
<212> DNA
<213> Sorghum bicolor
<400> 87
atggctcgaa ccaagcacca ggccgtgagg aagctgccgc agaagcccaa gaagaagctc 60
cagttcgagc gcgcaggtgg ggcgagtacg tcggcgaccc cggagaggag gaatgctggg 120
accgggggag gagccgcggc tcgcgttgca cgggggcgtg tggagaagaa gcatcgctgg 180
cgggcaggga ctgtagcgct gcgggagatc aggaagtacc agaagtccac tgagccgctc 240
atcccctttg cgcccttcgt acgtgtggtc aaagagttaa ctgtattcat aacagactgg 300
aggatagggc gctacacccc tgaagccctc cttgcgctgc aagaggcagc agaattccac 360
ttgatagaac tgtttgaagt ggcgaatctg tgtgccatcc atgccaagcg cgtaacagtc 420
atgcaaaagg acatacaact tgcaaggcgt atcggaggaa ggcgttggtc g 471
<210> 88
<211> 157
<212> PRT
<213> Sorghum bicolor
<400> 88
Met Ala Arg Thr Lys His Gln Ala Val Arg Lys Leu Pro Gln Lys Pro
1 5 10 15
Lys Lys Lys Leu Gln Phe Glu Arg Ala Gly Gly Ala Ser Thr Ser Ala
20 25 30
Thr Pro Glu Arg Arg Asn Ala Gly Thr Gly Gly Gly Ala Ala Ala Arg
35 40 45
Val Ala Arg Gly Arg Val Glu Lys Lys His Arg Trp Arg Ala Gly Thr
50 55 60
Val Ala Leu Arg Glu Ile Arg Lys Tyr Gln Lys Ser Thr Glu Pro Leu
65 70 75 80
Ile Pro Phe Ala Pro Phe Val Arg Val Val Lys Glu Leu Thr Val Phe
85 90 95
Ile Thr Asp Trp Arg Ile Gly Arg Tyr Thr Pro Glu Ala Leu Leu Ala
100 105 110
Leu Gln Glu Ala Ala Glu Phe His Leu Ile Glu Leu Phe Glu Val Ala
115 120 125
Asn Leu Cys Ala Ile His Ala Lys Arg Val Thr Val Met Gln Lys Asp
130 135 140
Ile Gln Leu Ala Arg Arg Ile Gly Gly Arg Arg Trp Ser
145 150 155
<210> 89
<211> 8400
<212> DNA
<213> Beta vulgaris
<400> 89
ctactctttc tctctctctc tctctccatt tctgtttgaa atcatgagag ttaaacacac 60
tgctgccagg aaatcaacca ccaacggtcc tcgttcaagt tagtttcctc tctcttcttc 120
ttttttgttc gcattctctc aatctatatt tcaaatttga aaaaaattgt gatgctcata 180
aaccctaaaa ttttcttgta cagaggctca gaaatctccg cgcagtttgc aatcaccaca 240
atcggttctc tctttgtact tttgatttgt ttttccttca tttgttcgat gaatggctct 300
taattgtctt ttatttactt gaaaattgca gccttctagt agttcaaagc gcaaatcact 360
cagaaacact gatgcaactc ctcaaagtaa ctttttcttt aatattaggt ttaattttac 420
tgctgtttgc caaattctgt tgaaattgta aaatattttt tttcttaaat ttgacggttt 480
cagagaagaa ggcttaccgc cgtaagccgg gcactgtggc actctgggaa atacgcaaat 540
ttcagaagtc attcaagccc ttgattcctg ctgcgccttt cattcgaaca gtatgtattt 600
tttttgtttg tacttaataa atgaattttg gactggtgtt tgtgtggctg catagaaata 660
tatttccata caactgaaat tgtcctagga ggtatcgatg aatgtttgct acaaaataaa 720
taaatataag tgattatatc ttgttaaaaa gccattataa ttgcaactta tatgtatgtt 780
gtaatgaggt caactagcta ttttgtgcaa agtcacccac actttaacat aattttgtgc 840
tctcgtaacc ttaaaaaaat ataagtaaag ggttgatttg gtctaattag agctgatgaa 900
acccaattag attgaaacat aaggtgaaat caggtggtga tcagcttcaa ttagatctaa 960
taagtgcagt ttagtttagc ttcggtgaaa tgaacacacc cttaaagata gaaaatcgac 1020
actatatatg gtccttttta gatatgatag ttcgatattc tgttttgggg tgtgttgaat 1080
gattaaatgg agtggtgaat agctgatggg aactagagaa gatgctcagt agacagttat 1140
tgtggagact atattactga ttacccctgt ttctgagtgg ttaggacaat gtgacaattg 1200
attttgggta ttatttgtag atgtttttct ttttgttaaa agtgccaaga taggtgtgca 1260
gttgctgatt ctcagtttgc taagaattag ctgtgtctgt atttcgtacc tcagttgatt 1320
ctaagtgaac atttctttga attgatgctt tgttcttgca tcatgcaact tggtgaagct 1380
ttcttgtagt tgctccagtg gcaatctagt ctggtatgtt tagaactctt gtgatggtat 1440
gagttcatca agatattggt gatccaatta gcctaaccaa tgttttttac cccctattgt 1500
cactgactta tactccctta tctataaaaa taattgtgac attgatccat ctcctcacaa 1560
tcattaatat tatatgtact gaccatcttt acactctcaa cactgaatct aagtagggga 1620
attttgggaa attcaatgat gaactagtac acccttcttc ccaataatat tgttgacctt 1680
tttattttga tttgtcccat attgtcctct ttggtaattt aatgtatatt cacccaattt 1740
tcttttcaat acccactata ccaacatata attggttaat tcatttttat taattatatt 1800
tcctaagagc ttgttgtgta aacgtggatg aatttgtagg catggatgaa gtattgttat 1860
aatgaggtga caacattact taatttcgaa ctgagggaca gagggatatg atgataaaac 1920
aacttttgct tgcttcttaa actcagaaga tagggtttac accaagtggc atgtaaaagt 1980
cactagatga ttatctatta caagggcttg tacaatctga agtacgatag gatttgaagt 2040
taacaacatt catcgaaagc tcataacttg tccttatatc aatataagtt gctggcatgt 2100
gaaattgcgt tgcaagcatc catgagctag ctcaactatt aactattaaa ctttatattt 2160
ttgcttgatc tagtatgagt cctactattt agtttctcca tctaccttaa tatgtcgcat 2220
acaccaacta atcattatcg ctagaatcaa taaacaaagc tttctttcct taggtgtatt 2280
agtacctagc tcctgtaata ccaagagcac ccaaattggg aagaaaaagt agaattggct 2340
catatctcta atcctacatt gatcattgaa aaggacctta aggttctcat actgaaacat 2400
catctttttg agcaggatat ctacgtagac gacaagaaag actactttgg ttgcccgtgc 2460
atttgagtgc atcagacaac ttctttacca ctgtctaacg gcttgctttg gccatattgt 2520
ggtcttctat gccaaaatta atgatatttc ttggcaccgc gctaatgata ttactgaatg 2580
cggatatcgt acgattagaa tttattcaaa gtaggtagca attactagtt ttgagcattg 2640
agtttcaata attagtaaat taagtgctaa acttgtacat tttggctaca tgtatttgaa 2700
ttagaattgg tacgaggaaa tatagcaaca ttacgggcaa tgttcactca agtagaagcc 2760
attacatcaa atagtactag ttgaagtatt agttctcata atactaatca ttgtcattaa 2820
tggaatattg gaacgtaaat gcctttaagg tgctgtagtt ttagtagaaa ttctactatt 2880
ctagtatgat aatgcaattt attgaaactg tttgtaagat agcttggatc ccacatcagt 2940
cttgatgcta aataaatgga tgtccataat cttctaatct ttaatttgtg tctcttacca 3000
aacgagaaaa aataggagaa atccaatttg catgacctca ataaggaaat gttgttaatg 3060
tgtgatgctt gtttctcatt tatagtctag agagagttat catgtccaag attgcagtct 3120
tggtactgag aaagtttgat tgttggttgg ctgcttcttg agcctctttt ttttagagta 3180
agacacttcc tagatataat tttctttatt tttttgtaaa ttccatatat actactacat 3240
taacaggttt aagtttatat taggtgagag agattactca ccagtttgct ccttatgttg 3300
gtcgttggca agctgaagct cagatggccc ttcaagaggt gcagaccaac tcttttagcc 3360
tttttttttc tggcatgtca agtgtggcta ttagattttc tgtgtgattc tcactcccat 3420
atatctatat atgtacatat taaagcacat tgatacctat cttgtcagat gtggtctttt 3480
caattctttt ctaagttgag attcttctct tggtcgtaga tatgctcctg ccgaaatata 3540
ctgctgtctt gttatccatc atgacttggt tatgcttgta tctgggcatt atcttggcat 3600
gcttaaaaac aagtattgaa cgagcctcct attgataaat tttactatta atattggatg 3660
gcttctcaaa ttctaatggc agtgagatac tgttaagttg ggagaaatag attaagaaac 3720
agaaagatgt ttaccatgag agcaattgaa atagaaaata gagtaacttt ttgcaaagat 3780
tttggtcctt tagattgttg aatactacct gataatgaag cattttctaa atttatgtgc 3840
tttctatcta tcagatactg gaatacaatc aaattcctat cacgtactga gcattgtgat 3900
cagattcttg cttgcttcct atcacatact ggaatccttt tgattgttga atacaaagat 3960
aatgaagcat tctctaactt tatgtgctgt aactacttat aatgattctt gcttgcctcc 4020
tatcacgtac tcaaatcctt tgtttgattt gtctcttata agaggaactt cctgtctttc 4080
tttgtcatga cttagtattt atagaggtgc caacttatgg ccttgacaac tgaagctttt 4140
atgcaaactc cggattttgt tgatggaagt acaagtaaca ctttagcatg tggattcagg 4200
tctaacggtt aagacttttt aatgaatgtt ttaactgtag tagtttattg atataaaaaa 4260
agtggtctct caaacttttt atgagatcat atcgaagtaa tcaaatttat gattcaggtg 4320
cttctgctat tattcttggt taagcatgtg ctatttttga cagtctgtca attgtaggct 4380
gcagagaatt ttattgtccg tttgtttgaa gatggtatgc tttgtgcaat tcatgccaaa 4440
cgagttacac tcagtgagta tctgatttcc ttcggtggtg ctgctattat gcattatata 4500
cactttgcct caatatcgtt atataaggag tccttgtttt catatttgtt tgatgcatat 4560
gttatatcct gtttagtggc tgctgcagtt gtgaacttac ggcctgtttg attagtggtc 4620
ataaatgatg gtaatactaa tataatttag tataaatttg taaaaaaaat gctaatatca 4680
atatttatgg taatgaaatt ttatcataaa acatgagttc tcttttataa gttttcatta 4740
ctatccaata ccaccttccc aagtggtaat gaacggtaat gaaattttag gaagaaaatg 4800
gatatttggg gattagatag cattaccatg ggtaatgaca tgagattttc tttacaactt 4860
tatactacga tgcattatca ttaccaccat ttatgaccca taaccaaaaa aaccataatg 4920
tgttaggttc atttttcatt tttctaataa tttgcttcat gaattttttc tggagatatc 4980
ttatctagat atttcttgcc aacatgtttc acctgataat tgatcgattt aatagttcag 5040
aactttccaa aaactatgct gctcggtgtt ggctgtcatc catcagttta agaaaactat 5100
tgacatgatt taagcctcgt cctgtactac taggaagggt aaactattgt tgcttccaaa 5160
aatgtctttt aagggcgtgt tcagcaacaa tagttgtagt agtagctttt agctgttagt 5220
tgtgctcgta gctgttagtg gttagtgtgt aactgttagc tgttcaagta gcggtataag 5280
atattgatgt tcggtaaaag aagctgtcaa aatagctgtt tacaaagaat taataaaaaa 5340
ctcaaacaaa gctttaatat ataatttatg caccactaaa gctaccccaa aagctacaaa 5400
ttgtagcttt ttacaaacac tactaaaaca ctacttgtaa cactaaaagc tacttatact 5460
actattttgc caaacattat tattttttct taattagtgt tttgacctag tcaagacact 5520
aaaagctact tgaaaagctt ttgccgaaca cgcccttagt agacaagagg ggggaggggg 5580
tcatcaagaa aatatgatta tactctcaac aaaaaaaaaa tgtaacttaa aaaaaataaa 5640
aataaataat tgactacttc aattaagaaa agaatagaat aaaaacatta cagtggatgt 5700
ctcatccaca tccctaattt aatggcacaa tagaataatt gttttaaatt ttagaaatta 5760
caacacaaga tgtaaattac tcttatcttc ctcttcgtaa tctttttact cttcctttac 5820
ctcttccttt acctctacat aaaatagaga attagagatt gattaagata attataagat 5880
tttagaaaca ttggttaaga aattcttcaa caaacataat caagtaactc cattatttta 5940
gtttagtgac ttgctattta tcaccctaat ttcaccatct accgccctcc ttggacaata 6000
ttgccccttc cactttcttc actcttcctt cctcacgcat cttatcatct ccttccacta 6060
tcacctttaa aaaagtgtgt caggcacaac aaaaacgctt ttatcaaccc acgcgaggcg 6120
aagtacgtca ggcgcaacaa ggcgcgcacc taattctgtc ttttgccaag gctgatggtg 6180
cacttagttt taaaaagcgc agcaaagatg tgcctaggcg caaggcggtg aaaaaattgc 6240
atccgtcagc agcggagtag aggctcacaa caataggtgc gaagaggcgt gcacgtacaa 6300
aagaagcaaa aataagaaac tcaaatatga gacccagtgt ttaacatgta aattcgatac 6360
ccagtgttta acatgtaaat tcgattaaaa gcccttaatt aattgcatga aattaattca 6420
ttttaaccta tactaaaagc cctaatatta gaaaatccta gtttgcaggt tgaggaattt 6480
ggaaaattga tgattgttgg atttgaaaaa attgttgccg gcgatgaatg tgaggtggtt 6540
tatggcacta gagaggttgg cgttcgttgc cgatgaagct ttccaaggtc attctctcct 6600
tgtcttcttc ctatgcctag ctctcttccc tctccttaat cttctcttct tttctattct 6660
ctctctttat cactacatta tgtttatttc tcgttcttcc cctatgtctt tcacttggac 6720
acttcggggg tatcttcatc ttttatctgc aatttgaagt ttgagaagct tccagagtcg 6780
agtgttaaac ttttgcttct ttttttttaa tcttttgccc ttttttctta gtggcccttg 6840
actagtgatg cacatgtgac caattactaa atgagctttt attttgtctc tcttcttttt 6900
caagcaattt tttttaagta aatcatctaa aacaaagtac tatccatttt agttgtgtaa 6960
atggtgctat tttaaaaccg cacaaaaatt aaaaacataa aaataaaggt gtgcttcgca 7020
tacaagatgt atgcgccttc gtcttgcccc ttttgagact aagactacca taagaactta 7080
gtcacttgag aatggaatgg gtgcaagatg gacgacgata attctaaaga cctctagaag 7140
gatagtgtat agtaactaat acgaaccgaa atataagttt aactaaaatt ttaaagtcta 7200
tatttccata tggtatatgc tggaatacac gaaatgtcca gaatttgtag tggaccacga 7260
tccacacgtc ttttcaggat tctaggtgta ttccaacgaa aaatataaga aaaccatatt 7320
ctactatctg gttgttgtca tcctttcctt gccggcgtga cttctcatcc ttttattttt 7380
gtccggtgct ggtgacacac tttcctatga tagtgtggtg caaagtaagg tgatgatatg 7440
gtgttttgta gaggtgtggt gatttttgtg gtggtgggtg gaagaggggt ggttgcatat 7500
agaaaggggt aagagtcaat gaggggtgga aggggacaag gggtatattg gtaaatgcat 7560
gtaacattag ggtggtgttg agtaattttt gggaagttaa tataaactac ccccttttgg 7620
tacaagagag aatacccgaa ctactgctct gatatttttg ttcacgttat ttgatgtaat 7680
tacgcaatta atttgttttc tataagcttc cgcacacaat tgtgcatata aggctagtct 7740
aatatgagac accaacataa ctgactttct tttgcaacga aggtaccttg tcagatttag 7800
aacatagcat caggatttta tttgttgtat ctgtcatcct tgtttattgc tttaattatg 7860
ctttgtatga tgcattttac cacttcgtat gaaaaaaagt gaaatttcat ttagtggtca 7920
tttacatatt acgagttgtg gacatgtttg aacatttgat tttggaaatt ttaagcctca 7980
tattatggag atttattgga cacaaatata gccataattc tccatcaact tgtttctaga 8040
agtgttgctc ttcctgatgt acttgaattc taattaggtt ttatcagaca ttatattata 8100
atgatatgat ttacaatttg ttgtagtgaa aaaggatttg gagctcgcgc gaaggattgg 8160
gggcagagag aggggatggt aactaaacaa cacagatgac tcatttattt aagggccaac 8220
aattgaattc gctgttgatt tcatctgtat atactgctct aggcttctat tccaatgtaa 8280
tttataaatc caaggttagt agcatgttaa gctttgtatt cagtataatg agacttatat 8340
tttgcagttg agattttagt tgtttgatgt gacttgtaaa ttgtaacttg taagtgacgt 8400
<210> 90
<211> 462
<212> DNA
<213> Beta vulgaris
<400> 90
atgagagtta aacacactgc tgccaggaaa tcaaccacca atggtcctcg ttcaaaggct 60
cagaaatctc cgcgcagttt gcaatcacca caatcgcctt ctagtagttc aaagcgcaaa 120
tcacgcagaa acactgatgc aactcctcaa aagaagaagg cttaccgccg taagccgggc 180
actgtggcac tctgggaaat acgcaaattt cagaagtcat tcaagccctt gattcctgct 240
gcgcctttca ttcgaacagt gagagagatt actcaccagt ttgctcctta tgttggtcgt 300
tggcaagctg aagctcagat ggcccttcaa gaggctgcag agaattttat tgtccgtttg 360
tttgaagatg gtatgctttg tgcaattcat gccaaacgag ttacactcat gaaaaaggat 420
ttggagctcg cgcgaaggat tgggggcaga gagaggggat gg 462
<210> 91
<211> 154
<212> PRT
<213> Beta vulgaris
<400> 91
Met Arg Val Lys His Thr Ala Ala Arg Lys Ser Thr Thr Asn Gly Pro
1 5 10 15
Arg Ser Lys Ala Gln Lys Ser Pro Arg Ser Leu Gln Ser Pro Gln Ser
20 25 30
Pro Ser Ser Ser Ser Lys Arg Lys Ser Arg Arg Asn Thr Asp Ala Thr
35 40 45
Pro Gln Lys Lys Lys Ala Tyr Arg Arg Lys Pro Gly Thr Val Ala Leu
50 55 60
Trp Glu Ile Arg Lys Phe Gln Lys Ser Phe Lys Pro Leu Ile Pro Ala
65 70 75 80
Ala Pro Phe Ile Arg Thr Val Arg Glu Ile Thr His Gln Phe Ala Pro
85 90 95
Tyr Val Gly Arg Trp Gln Ala Glu Ala Gln Met Ala Leu Gln Glu Ala
100 105 110
Ala Glu Asn Phe Ile Val Arg Leu Phe Glu Asp Gly Met Leu Cys Ala
115 120 125
Ile His Ala Lys Arg Val Thr Leu Met Lys Lys Asp Leu Glu Leu Ala
130 135 140
Arg Arg Ile Gly Gly Arg Glu Arg Gly Trp
145 150
<210> 92
<211> 8400
<212> DNA
<213> Beta vulgaris
<400> 92
ctactctttc tctctctctc tctctccatt tctgtttgaa atcatgagag ttaaacacac 60
tgctgccagg aaatcaacca ccaacggtcc tcgttcaagt tagtttcctc tctcttcttc 120
ttttttgttc gcattctctc aatctatatt tcaaatttga aaaaaattgt gatgctcata 180
aaccctaaaa ttttcttgta cagaggctca gaaatctccg cgcagtttgc aatcaccaca 240
atcggttctc tctttgtact tttgatttgt ttttccttca tttgttcgat gaatggctct 300
taattgtctt ttatttactt gaaaattgca gccttctagt agttcaaagc gcaaatcact 360
cagaaacact gatgcaactc ctcaaagtaa ctttttcttt aatattaggt ttaattttac 420
tgctgtttgc caaattctgt tgaaattgta aaatattttt tttcttaaat ttgacggttt 480
cagagaagaa ggcttaccgc cgtaagccgg gcactgtggc actctgggaa atacgcaaat 540
ttcagaagtc attcaagccc ttgattcctg ctgcgccttt cattcgaaca gtatgtattt 600
tttttgtttg tacttaataa atgaattttg gactggtgtt tgtgtggctg catagaaata 660
tatttccata caactgaaat tgtcctagga ggtatcgatg aatgtttgct acaaaataaa 720
taaatataag tgattatatc ttgttaaaaa gccattataa ttgcaactta tatgtatgtt 780
gtaatgaggt caactagcta ttttgtgcaa agtcacccac actttaacat aattttgtgc 840
tctcgtaacc ttaaaaaaat ataagtaaag ggttgatttg gtctaattag agctgatgaa 900
acccaattag attgaaacat aaggtgaaat caggtggtga tcagcttcaa ttagatctaa 960
taagtgcagt ttagtttagc ttcggtgaaa tgaacacacc cttaaagata gaaaatcgac 1020
actatatatg gtccttttta gatatgatag ttcgatattc tgttttgggg tgtgttgaat 1080
gattaaatgg agtggtgaat agctgatggg aactagagaa gatgctcagt agacagttat 1140
tgtggagact atattactga ttacccctgt ttctgagtgg ttaggacaat gtgacaattg 1200
attttgggta ttatttgtag atgtttttct ttttgttaaa agtgccaaga taggtgtgca 1260
gttgctgatt ctcagtttgc taagaattag ctgtgtctgt atttcgtacc tcagttgatt 1320
ctaagtgaac atttctttga attgatgctt tgttcttgca tcatgcaact tggtgaagct 1380
ttcttgtagt tgctccagtg gcaatctagt ctggtatgtt tagaactctt gtgatggtat 1440
gagttcatca agatattggt gatccaatta gcctaaccaa tgttttttac cccctattgt 1500
cactgactta tactccctta tctataaaaa taattgtgac attgatccat ctcctcacaa 1560
tcattaatat tatatgtact gaccatcttt acactctcaa cactgaatct aagtagggga 1620
attttgggaa attcaatgat gaactagtac acccttcttc ccaataatat tgttgacctt 1680
tttattttga tttgtcccat attgtcctct ttggtaattt aatgtatatt cacccaattt 1740
tcttttcaat acccactata ccaacatata attggttaat tcatttttat taattatatt 1800
tcctaagagc ttgttgtgta aacgtggatg aatttgtagg catggatgaa gtattgttat 1860
aatgaggtga caacattact taatttcgaa ctgagggaca gagggatatg atgataaaac 1920
aacttttgct tgcttcttaa actcagaaga tagggtttac accaagtggc atgtaaaagt 1980
cactagatga ttatctatta caagggcttg tacaatctga agtacgatag gatttgaagt 2040
taacaacatt catcgaaagc tcataacttg tccttatatc aatataagtt gctggcatgt 2100
gaaattgcgt tgcaagcatc catgagctag ctcaactatt aactattaaa ctttatattt 2160
ttgcttgatc tagtatgagt cctactattt agtttctcca tctaccttaa tatgtcgcat 2220
acaccaacta atcattatcg ctagaatcaa taaacaaagc tttctttcct taggtgtatt 2280
agtacctagc tcctgtaata ccaagagcac ccaaattggg aagaaaaagt agaattggct 2340
catatctcta atcctacatt gatcattgaa aaggacctta aggttctcat actgaaacat 2400
catctttttg agcaggatat ctacgtagac gacaagaaag actactttgg ttgcccgtgc 2460
atttgagtgc atcagacaac ttctttacca ctgtctaacg gcttgctttg gccatattgt 2520
ggtcttctat gccaaaatta atgatatttc ttggcaccgc gctaatgata ttactgaatg 2580
cggatatcgt acgattagaa tttattcaaa gtaggtagca attactagtt ttgagcattg 2640
agtttcaata attagtaaat taagtgctaa acttgtacat tttggctaca tgtatttgaa 2700
ttagaattgg tacgaggaaa tatagcaaca ttacgggcaa tgttcactca agtagaagcc 2760
attacatcaa atagtactag ttgaagtatt agttctcata atactaatca ttgtcattaa 2820
tggaatattg gaacgtaaat gcctttaagg tgctgtagtt ttagtagaaa ttctactatt 2880
ctagtatgat aatgcaattt attgaaactg tttgtaagat agcttggatc ccacatcagt 2940
cttgatgcta aataaatgga tgtccataat cttctaatct ttaatttgtg tctcttacca 3000
aacgagaaaa aataggagaa atccaatttg catgacctca ataaggaaat gttgttaatg 3060
tgtgatgctt gtttctcatt tatagtctag agagagttat catgtccaag attgcagtct 3120
tggtactgag aaagtttgat tgttggttgg ctgcttcttg agcctctttt ttttagagta 3180
agacacttcc tagatataat tttctttatt tttttgtaaa ttccatatat actactacat 3240
taacaggttt aagtttatat taggtgagag agattactca ccagtttgct ccttatgttg 3300
gtcgttggca agctgaagct ctgatggccc ctcaagaggt gcagaccaac tcttttagcc 3360
tttttttttc tggcatgtca agtgtggcta ttagattttc tgtgtgattc tcactcccat 3420
atatctatat atgtacatat taaagcacat tgatacctat cttgtcagat gtggtctttt 3480
caattctttt ctaagttgag attcttctct tggtcgtaga tatgctcctg ccgaaatata 3540
ctgctgtctt gttatccatc atgacttggt tatgcttgta tctgggcatt atcttggcat 3600
gcttaaaaac aagtattgaa cgagcctcct attgataaat tttactatta atattggatg 3660
gcttctcaaa ttctaatggc agtgagatac tgttaagttg ggagaaatag attaagaaac 3720
agaaagatgt ttaccatgag agcaattgaa atagaaaata gagtaacttt ttgcaaagat 3780
tttggtcctt tagattgttg aatactacct gataatgaag cattttctaa atttatgtgc 3840
tttctatcta tcagatactg gaatacaatc aaattcctat cacgtactga gcattgtgat 3900
cagattcttg cttgcttcct atcacatact ggaatccttt tgattgttga atacaaagat 3960
aatgaagcat tctctaactt tatgtgctgt aactacttat aatgattctt gcttgcctcc 4020
tatcacgtac tcaaatcctt tgtttgattt gtctcttata agaggaactt cctgtctttc 4080
tttgtcatga cttagtattt atagaggtgc caacttatgg ccttgacaac tgaagctttt 4140
atgcaaactc cggattttgt tgatggaagt acaagtaaca ctttagcatg tggattcagg 4200
tctaacggtt aagacttttt aatgaatgtt ttaactgtag tagtttattg atataaaaaa 4260
agtggtctct caaacttttt atgagatcat atcgaagtaa tcaaatttat gattcaggtg 4320
cttctgctat tattcttggt taagcatgtg ctatttttga cagtctgtca attgtaggct 4380
gcagagaatt ttattgtccg tttgtttgaa gatggtatgc tttgtgcaat tcatgccaaa 4440
cgagttacac tcagtgagta tctgatttcc ttcggtggtg ctgctattat gcattatata 4500
cactttgcct caatatcgtt atataaggag tccttgtttt catatttgtt tgatgcatat 4560
gttatatcct gtttagtggc tgctgcagtt gtgaacttac ggcctgtttg attagtggtc 4620
ataaatgatg gtaatactaa tataatttag tataaatttg taaaaaaaat gctaatatca 4680
atatttatgg taatgaaatt ttatcataaa acatgagttc tcttttataa gttttcatta 4740
ctatccaata ccaccttccc aagtggtaat gaacggtaat gaaattttag gaagaaaatg 4800
gatatttggg gattagatag cattaccatg ggtaatgaca tgagattttc tttacaactt 4860
tatactacga tgcattatca ttaccaccat ttatgaccca taaccaaaaa aaccataatg 4920
tgttaggttc atttttcatt tttctaataa tttgcttcat gaattttttc tggagatatc 4980
ttatctagat atttcttgcc aacatgtttc acctgataat tgatcgattt aatagttcag 5040
aactttccaa aaactatgct gctcggtgtt ggctgtcatc catcagttta agaaaactat 5100
tgacatgatt taagcctcgt cctgtactac taggaagggt aaactattgt tgcttccaaa 5160
aatgtctttt aagggcgtgt tcagcaacaa tagttgtagt agtagctttt agctgttagt 5220
tgtgctcgta gctgttagtg gttagtgtgt aactgttagc tgttcaagta gcggtataag 5280
atattgatgt tcggtaaaag aagctgtcaa aatagctgtt tacaaagaat taataaaaaa 5340
ctcaaacaaa gctttaatat ataatttatg caccactaaa gctaccccaa aagctacaaa 5400
ttgtagcttt ttacaaacac tactaaaaca ctacttgtaa cactaaaagc tacttatact 5460
actattttgc caaacattat tattttttct taattagtgt tttgacctag tcaagacact 5520
aaaagctact tgaaaagctt ttgccgaaca cgcccttagt agacaagagg ggggaggggg 5580
tcatcaagaa aatatgatta tactctcaac aaaaaaaaaa tgtaacttaa aaaaaataaa 5640
aataaataat tgactacttc aattaagaaa agaatagaat aaaaacatta cagtggatgt 5700
ctcatccaca tccctaattt aatggcacaa tagaataatt gttttaaatt ttagaaatta 5760
caacacaaga tgtaaattac tcttatcttc ctcttcgtaa tctttttact cttcctttac 5820
ctcttccttt acctctacat aaaatagaga attagagatt gattaagata attataagat 5880
tttagaaaca ttggttaaga aattcttcaa caaacataat caagtaactc cattatttta 5940
gtttagtgac ttgctattta tcaccctaat ttcaccatct accgccctcc ttggacaata 6000
ttgccccttc cactttcttc actcttcctt cctcacgcat cttatcatct ccttccacta 6060
tcacctttaa aaaagtgtgt caggcacaac aaaaacgctt ttatcaaccc acgcgaggcg 6120
aagtacgtca ggcgcaacaa ggcgcgcacc taattctgtc ttttgccaag gctgatggtg 6180
cacttagttt taaaaagcgc agcaaagatg tgcctaggcg caaggcggtg aaaaaattgc 6240
atccgtcagc agcggagtag aggctcacaa caataggtgc gaagaggcgt gcacgtacaa 6300
aagaagcaaa aataagaaac tcaaatatga gacccagtgt ttaacatgta aattcgatac 6360
ccagtgttta acatgtaaat tcgattaaaa gcccttaatt aattgcatga aattaattca 6420
ttttaaccta tactaaaagc cctaatatta gaaaatccta gtttgcaggt tgaggaattt 6480
ggaaaattga tgattgttgg atttgaaaaa attgttgccg gcgatgaatg tgaggtggtt 6540
tatggcacta gagaggttgg cgttcgttgc cgatgaagct ttccaaggtc attctctcct 6600
tgtcttcttc ctatgcctag ctctcttccc tctccttaat cttctcttct tttctattct 6660
ctctctttat cactacatta tgtttatttc tcgttcttcc cctatgtctt tcacttggac 6720
acttcggggg tatcttcatc ttttatctgc aatttgaagt ttgagaagct tccagagtcg 6780
agtgttaaac ttttgcttct ttttttttaa tcttttgccc ttttttctta gtggcccttg 6840
actagtgatg cacatgtgac caattactaa atgagctttt attttgtctc tcttcttttt 6900
caagcaattt tttttaagta aatcatctaa aacaaagtac tatccatttt agttgtgtaa 6960
atggtgctat tttaaaaccg cacaaaaatt aaaaacataa aaataaaggt gtgcttcgca 7020
tacaagatgt atgcgccttc gtcttgcccc ttttgagact aagactacca taagaactta 7080
gtcacttgag aatggaatgg gtgcaagatg gacgacgata attctaaaga cctctagaag 7140
gatagtgtat agtaactaat acgaaccgaa atataagttt aactaaaatt ttaaagtcta 7200
tatttccata tggtatatgc tggaatacac gaaatgtcca gaatttgtag tggaccacga 7260
tccacacgtc ttttcaggat tctaggtgta ttccaacgaa aaatataaga aaaccatatt 7320
ctactatctg gttgttgtca tcctttcctt gccggcgtga cttctcatcc ttttattttt 7380
gtccggtgct ggtgacacac tttcctatga tagtgtggtg caaagtaagg tgatgatatg 7440
gtgttttgta gaggtgtggt gatttttgtg gtggtgggtg gaagaggggt ggttgcatat 7500
agaaaggggt aagagtcaat gaggggtgga aggggacaag gggtatattg gtaaatgcat 7560
gtaacattag ggtggtgttg agtaattttt gggaagttaa tataaactac ccccttttgg 7620
tacaagagag aatacccgaa ctactgctct gatatttttg ttcacgttat ttgatgtaat 7680
tacgcaatta atttgttttc tataagcttc cgcacacaat tgtgcatata aggctagtct 7740
aatatgagac accaacataa ctgactttct tttgcaacga aggtaccttg tcagatttag 7800
aacatagcat caggatttta tttgttgtat ctgtcatcct tgtttattgc tttaattatg 7860
ctttgtatga tgcattttac cacttcgtat gaaaaaaagt gaaatttcat ttagtggtca 7920
tttacatatt acgagttgtg gacatgtttg aacatttgat tttggaaatt ttaagcctca 7980
tattatggag atttattgga cacaaatata gccataattc tccatcaact tgtttctaga 8040
agtgttgctc ttcctgatgt acttgaattc taattaggtt ttatcagaca ttatattata 8100
atgatatgat ttacaatttg ttgtagtgaa aaaggatttg gagctcgcgc gaaggattgg 8160
gggcagagag aggggatggt aactaaacaa cacagatgac tcatttattt aagggccaac 8220
aattgaattc gctgttgatt tcatctgtat atactgctct aggcttctat tccaatgtaa 8280
tttataaatc caaggttagt agcatgttaa gctttgtatt cagtataatg agacttatat 8340
tttgcagttg agattttagt tgtttgatgt gacttgtaaa ttgtaacttg taagtgacgt 8400
<210> 93
<211> 462
<212> DNA
<213> Beta vulgaris
<400> 93
atgagagtta aacacactgc tgccaggaaa tcaaccacca atggtcctcg ttcaaaggct 60
cagaaatctc cgcgcagttt gcaatcacca caatcgcctt ctagtagttc aaagcgcaaa 120
tcacgcagaa acactgatgc aactcctcaa aagaagaagg cttaccgccg taagccgggc 180
actgtggcac tctgggaaat acgcaaattt cagaagtcat tcaagccctt gattcctgct 240
gcgcctttca ttcgaacagt gagagagatt actcaccagt ttgctcctta tgttggtcgt 300
tggcaagctg aagctctgat ggcccctcaa gaggctgcag agaattttat tgtccgtttg 360
tttgaagatg gtatgctttg tgcaattcat gccaaacgag ttacactcat gaaaaaggat 420
ttggagctcg cgcgaaggat tgggggcaga gagaggggat gg 462
<210> 94
<211> 154
<212> PRT
<213> Beta vulgaris
<400> 94
Met Arg Val Lys His Thr Ala Ala Arg Lys Ser Thr Thr Asn Gly Pro
1 5 10 15
Arg Ser Lys Ala Gln Lys Ser Pro Arg Ser Leu Gln Ser Pro Gln Ser
20 25 30
Pro Ser Ser Ser Ser Lys Arg Lys Ser Arg Arg Asn Thr Asp Ala Thr
35 40 45
Pro Gln Lys Lys Lys Ala Tyr Arg Arg Lys Pro Gly Thr Val Ala Leu
50 55 60
Trp Glu Ile Arg Lys Phe Gln Lys Ser Phe Lys Pro Leu Ile Pro Ala
65 70 75 80
Ala Pro Phe Ile Arg Thr Val Arg Glu Ile Thr His Gln Phe Ala Pro
85 90 95
Tyr Val Gly Arg Trp Gln Ala Glu Ala Leu Met Ala Pro Gln Glu Ala
100 105 110
Ala Glu Asn Phe Ile Val Arg Leu Phe Glu Asp Gly Met Leu Cys Ala
115 120 125
Ile His Ala Lys Arg Val Thr Leu Met Lys Lys Asp Leu Glu Leu Ala
130 135 140
Arg Arg Ile Gly Gly Arg Glu Arg Gly Trp
145 150
<210> 95
<211> 8400
<212> DNA
<213> Beta vulgaris
<400> 95
ctactctttc tctctctctc tctctccatt tctgtttgaa atcatgagag ttaaacacac 60
tgctgccagg aaatcaacca ccaacggtcc tcgttcaagt tagtttcctc tctcttcttc 120
ttttttgttc gcattctctc aatctatatt tcaaatttga aaaaaattgt gatgctcata 180
aaccctaaaa ttttcttgta cagaggctca gaaatctccg cgcagtttgc aatcaccaca 240
atcggttctc tctttgtact tttgatttgt ttttccttca tttgttcgat gaatggctct 300
taattgtctt ttatttactt gaaaattgca gccttctagt agttcaaagc gcaaatcact 360
cagaaacact gatgcaactc ctcaaagtaa ctttttcttt aatattaggt ttaattttac 420
tgctgtttgc caaattctgt tgaaattgta aaatattttt tttcttaaat ttgacggttt 480
cagagaagaa ggcttaccgc cgtaagccgg gcactgtggc actctgggaa atacgcaaat 540
ttcagaagtc attcaagccc ttgattcctg ctgcgccttt cattcgaaca gtatgtattt 600
tttttgtttg tacttaataa atgaattttg gactggtgtt tgtgtggctg catagaaata 660
tatttccata caactgaaat tgtcctagga ggtatcgatg aatgtttgct acaaaataaa 720
taaatataag tgattatatc ttgttaaaaa gccattataa ttgcaactta tatgtatgtt 780
gtaatgaggt caactagcta ttttgtgcaa agtcacccac actttaacat aattttgtgc 840
tctcgtaacc ttaaaaaaat ataagtaaag ggttgatttg gtctaattag agctgatgaa 900
acccaattag attgaaacat aaggtgaaat caggtggtga tcagcttcaa ttagatctaa 960
taagtgcagt ttagtttagc ttcggtgaaa tgaacacacc cttaaagata gaaaatcgac 1020
actatatatg gtccttttta gatatgatag ttcgatattc tgttttgggg tgtgttgaat 1080
gattaaatgg agtggtgaat agctgatggg aactagagaa gatgctcagt agacagttat 1140
tgtggagact atattactga ttacccctgt ttctgagtgg ttaggacaat gtgacaattg 1200
attttgggta ttatttgtag atgtttttct ttttgttaaa agtgccaaga taggtgtgca 1260
gttgctgatt ctcagtttgc taagaattag ctgtgtctgt atttcgtacc tcagttgatt 1320
ctaagtgaac atttctttga attgatgctt tgttcttgca tcatgcaact tggtgaagct 1380
ttcttgtagt tgctccagtg gcaatctagt ctggtatgtt tagaactctt gtgatggtat 1440
gagttcatca agatattggt gatccaatta gcctaaccaa tgttttttac cccctattgt 1500
cactgactta tactccctta tctataaaaa taattgtgac attgatccat ctcctcacaa 1560
tcattaatat tatatgtact gaccatcttt acactctcaa cactgaatct aagtagggga 1620
attttgggaa attcaatgat gaactagtac acccttcttc ccaataatat tgttgacctt 1680
tttattttga tttgtcccat attgtcctct ttggtaattt aatgtatatt cacccaattt 1740
tcttttcaat acccactata ccaacatata attggttaat tcatttttat taattatatt 1800
tcctaagagc ttgttgtgta aacgtggatg aatttgtagg catggatgaa gtattgttat 1860
aatgaggtga caacattact taatttcgaa ctgagggaca gagggatatg atgataaaac 1920
aacttttgct tgcttcttaa actcagaaga tagggtttac accaagtggc atgtaaaagt 1980
cactagatga ttatctatta caagggcttg tacaatctga agtacgatag gatttgaagt 2040
taacaacatt catcgaaagc tcataacttg tccttatatc aatataagtt gctggcatgt 2100
gaaattgcgt tgcaagcatc catgagctag ctcaactatt aactattaaa ctttatattt 2160
ttgcttgatc tagtatgagt cctactattt agtttctcca tctaccttaa tatgtcgcat 2220
acaccaacta atcattatcg ctagaatcaa taaacaaagc tttctttcct taggtgtatt 2280
agtacctagc tcctgtaata ccaagagcac ccaaattggg aagaaaaagt agaattggct 2340
catatctcta atcctacatt gatcattgaa aaggacctta aggttctcat actgaaacat 2400
catctttttg agcaggatat ctacgtagac gacaagaaag actactttgg ttgcccgtgc 2460
atttgagtgc atcagacaac ttctttacca ctgtctaacg gcttgctttg gccatattgt 2520
ggtcttctat gccaaaatta atgatatttc ttggcaccgc gctaatgata ttactgaatg 2580
cggatatcgt acgattagaa tttattcaaa gtaggtagca attactagtt ttgagcattg 2640
agtttcaata attagtaaat taagtgctaa acttgtacat tttggctaca tgtatttgaa 2700
ttagaattgg tacgaggaaa tatagcaaca ttacgggcaa tgttcactca agtagaagcc 2760
attacatcaa atagtactag ttgaagtatt agttctcata atactaatca ttgtcattaa 2820
tggaatattg gaacgtaaat gcctttaagg tgctgtagtt ttagtagaaa ttctactatt 2880
ctagtatgat aatgcaattt attgaaactg tttgtaagat agcttggatc ccacatcagt 2940
cttgatgcta aataaatgga tgtccataat cttctaatct ttaatttgtg tctcttacca 3000
aacgagaaaa aataggagaa atccaatttg catgacctca ataaggaaat gttgttaatg 3060
tgtgatgctt gtttctcatt tatagtctag agagagttat catgtccaag attgcagtct 3120
tggtactgag aaagtttgat tgttggttgg ctgcttcttg agcctctttt ttttagagta 3180
agacacttcc tagatataat tttctttatt tttttgtaaa ttccatatat actactacat 3240
taacaggttt aagtttatat taggtgagag agattactca ccagtttgct ccttatgttg 3300
gtcgttggca agctgaagct ctgatggccc ttctagaggt gcagaccaac tcttttagcc 3360
tttttttttc tggcatgtca agtgtggcta ttagattttc tgtgtgattc tcactcccat 3420
atatctatat atgtacatat taaagcacat tgatacctat cttgtcagat gtggtctttt 3480
caattctttt ctaagttgag attcttctct tggtcgtaga tatgctcctg ccgaaatata 3540
ctgctgtctt gttatccatc atgacttggt tatgcttgta tctgggcatt atcttggcat 3600
gcttaaaaac aagtattgaa cgagcctcct attgataaat tttactatta atattggatg 3660
gcttctcaaa ttctaatggc agtgagatac tgttaagttg ggagaaatag attaagaaac 3720
agaaagatgt ttaccatgag agcaattgaa atagaaaata gagtaacttt ttgcaaagat 3780
tttggtcctt tagattgttg aatactacct gataatgaag cattttctaa atttatgtgc 3840
tttctatcta tcagatactg gaatacaatc aaattcctat cacgtactga gcattgtgat 3900
cagattcttg cttgcttcct atcacatact ggaatccttt tgattgttga atacaaagat 3960
aatgaagcat tctctaactt tatgtgctgt aactacttat aatgattctt gcttgcctcc 4020
tatcacgtac tcaaatcctt tgtttgattt gtctcttata agaggaactt cctgtctttc 4080
tttgtcatga cttagtattt atagaggtgc caacttatgg ccttgacaac tgaagctttt 4140
atgcaaactc cggattttgt tgatggaagt acaagtaaca ctttagcatg tggattcagg 4200
tctaacggtt aagacttttt aatgaatgtt ttaactgtag tagtttattg atataaaaaa 4260
agtggtctct caaacttttt atgagatcat atcgaagtaa tcaaatttat gattcaggtg 4320
cttctgctat tattcttggt taagcatgtg ctatttttga cagtctgtca attgtaggct 4380
gcagagaatt ttattgtccg tttgtttgaa gatggtatgc tttgtgcaat tcatgccaaa 4440
cgagttacac tcagtgagta tctgatttcc ttcggtggtg ctgctattat gcattatata 4500
cactttgcct caatatcgtt atataaggag tccttgtttt catatttgtt tgatgcatat 4560
gttatatcct gtttagtggc tgctgcagtt gtgaacttac ggcctgtttg attagtggtc 4620
ataaatgatg gtaatactaa tataatttag tataaatttg taaaaaaaat gctaatatca 4680
atatttatgg taatgaaatt ttatcataaa acatgagttc tcttttataa gttttcatta 4740
ctatccaata ccaccttccc aagtggtaat gaacggtaat gaaattttag gaagaaaatg 4800
gatatttggg gattagatag cattaccatg ggtaatgaca tgagattttc tttacaactt 4860
tatactacga tgcattatca ttaccaccat ttatgaccca taaccaaaaa aaccataatg 4920
tgttaggttc atttttcatt tttctaataa tttgcttcat gaattttttc tggagatatc 4980
ttatctagat atttcttgcc aacatgtttc acctgataat tgatcgattt aatagttcag 5040
aactttccaa aaactatgct gctcggtgtt ggctgtcatc catcagttta agaaaactat 5100
tgacatgatt taagcctcgt cctgtactac taggaagggt aaactattgt tgcttccaaa 5160
aatgtctttt aagggcgtgt tcagcaacaa tagttgtagt agtagctttt agctgttagt 5220
tgtgctcgta gctgttagtg gttagtgtgt aactgttagc tgttcaagta gcggtataag 5280
atattgatgt tcggtaaaag aagctgtcaa aatagctgtt tacaaagaat taataaaaaa 5340
ctcaaacaaa gctttaatat ataatttatg caccactaaa gctaccccaa aagctacaaa 5400
ttgtagcttt ttacaaacac tactaaaaca ctacttgtaa cactaaaagc tacttatact 5460
actattttgc caaacattat tattttttct taattagtgt tttgacctag tcaagacact 5520
aaaagctact tgaaaagctt ttgccgaaca cgcccttagt agacaagagg ggggaggggg 5580
tcatcaagaa aatatgatta tactctcaac aaaaaaaaaa tgtaacttaa aaaaaataaa 5640
aataaataat tgactacttc aattaagaaa agaatagaat aaaaacatta cagtggatgt 5700
ctcatccaca tccctaattt aatggcacaa tagaataatt gttttaaatt ttagaaatta 5760
caacacaaga tgtaaattac tcttatcttc ctcttcgtaa tctttttact cttcctttac 5820
ctcttccttt acctctacat aaaatagaga attagagatt gattaagata attataagat 5880
tttagaaaca ttggttaaga aattcttcaa caaacataat caagtaactc cattatttta 5940
gtttagtgac ttgctattta tcaccctaat ttcaccatct accgccctcc ttggacaata 6000
ttgccccttc cactttcttc actcttcctt cctcacgcat cttatcatct ccttccacta 6060
tcacctttaa aaaagtgtgt caggcacaac aaaaacgctt ttatcaaccc acgcgaggcg 6120
aagtacgtca ggcgcaacaa ggcgcgcacc taattctgtc ttttgccaag gctgatggtg 6180
cacttagttt taaaaagcgc agcaaagatg tgcctaggcg caaggcggtg aaaaaattgc 6240
atccgtcagc agcggagtag aggctcacaa caataggtgc gaagaggcgt gcacgtacaa 6300
aagaagcaaa aataagaaac tcaaatatga gacccagtgt ttaacatgta aattcgatac 6360
ccagtgttta acatgtaaat tcgattaaaa gcccttaatt aattgcatga aattaattca 6420
ttttaaccta tactaaaagc cctaatatta gaaaatccta gtttgcaggt tgaggaattt 6480
ggaaaattga tgattgttgg atttgaaaaa attgttgccg gcgatgaatg tgaggtggtt 6540
tatggcacta gagaggttgg cgttcgttgc cgatgaagct ttccaaggtc attctctcct 6600
tgtcttcttc ctatgcctag ctctcttccc tctccttaat cttctcttct tttctattct 6660
ctctctttat cactacatta tgtttatttc tcgttcttcc cctatgtctt tcacttggac 6720
acttcggggg tatcttcatc ttttatctgc aatttgaagt ttgagaagct tccagagtcg 6780
agtgttaaac ttttgcttct ttttttttaa tcttttgccc ttttttctta gtggcccttg 6840
actagtgatg cacatgtgac caattactaa atgagctttt attttgtctc tcttcttttt 6900
caagcaattt tttttaagta aatcatctaa aacaaagtac tatccatttt agttgtgtaa 6960
atggtgctat tttaaaaccg cacaaaaatt aaaaacataa aaataaaggt gtgcttcgca 7020
tacaagatgt atgcgccttc gtcttgcccc ttttgagact aagactacca taagaactta 7080
gtcacttgag aatggaatgg gtgcaagatg gacgacgata attctaaaga cctctagaag 7140
gatagtgtat agtaactaat acgaaccgaa atataagttt aactaaaatt ttaaagtcta 7200
tatttccata tggtatatgc tggaatacac gaaatgtcca gaatttgtag tggaccacga 7260
tccacacgtc ttttcaggat tctaggtgta ttccaacgaa aaatataaga aaaccatatt 7320
ctactatctg gttgttgtca tcctttcctt gccggcgtga cttctcatcc ttttattttt 7380
gtccggtgct ggtgacacac tttcctatga tagtgtggtg caaagtaagg tgatgatatg 7440
gtgttttgta gaggtgtggt gatttttgtg gtggtgggtg gaagaggggt ggttgcatat 7500
agaaaggggt aagagtcaat gaggggtgga aggggacaag gggtatattg gtaaatgcat 7560
gtaacattag ggtggtgttg agtaattttt gggaagttaa tataaactac ccccttttgg 7620
tacaagagag aatacccgaa ctactgctct gatatttttg ttcacgttat ttgatgtaat 7680
tacgcaatta atttgttttc tataagcttc cgcacacaat tgtgcatata aggctagtct 7740
aatatgagac accaacataa ctgactttct tttgcaacga aggtaccttg tcagatttag 7800
aacatagcat caggatttta tttgttgtat ctgtcatcct tgtttattgc tttaattatg 7860
ctttgtatga tgcattttac cacttcgtat gaaaaaaagt gaaatttcat ttagtggtca 7920
tttacatatt acgagttgtg gacatgtttg aacatttgat tttggaaatt ttaagcctca 7980
tattatggag atttattgga cacaaatata gccataattc tccatcaact tgtttctaga 8040
agtgttgctc ttcctgatgt acttgaattc taattaggtt ttatcagaca ttatattata 8100
atgatatgat ttacaatttg ttgtagtgaa aaaggatttg gagctcgcgc gaaggattgg 8160
gggcagagag aggggatggt aactaaacaa cacagatgac tcatttattt aagggccaac 8220
aattgaattc gctgttgatt tcatctgtat atactgctct aggcttctat tccaatgtaa 8280
tttataaatc caaggttagt agcatgttaa gctttgtatt cagtataatg agacttatat 8340
tttgcagttg agattttagt tgtttgatgt gacttgtaaa ttgtaacttg taagtgacgt 8400
<210> 96
<211> 462
<212> DNA
<213> Beta vulgaris
<400> 96
atgagagtta aacacactgc tgccaggaaa tcaaccacca atggtcctcg ttcaaaggct 60
cagaaatctc cgcgcagttt gcaatcacca caatcgcctt ctagtagttc aaagcgcaaa 120
tcacgcagaa acactgatgc aactcctcaa aagaagaagg cttaccgccg taagccgggc 180
actgtggcac tctgggaaat acgcaaattt cagaagtcat tcaagccctt gattcctgct 240
gcgcctttca ttcgaacagt gagagagatt actcaccagt ttgctcctta tgttggtcgt 300
tggcaagctg aagctctgat ggcccttcta gaggctgcag agaattttat tgtccgtttg 360
tttgaagatg gtatgctttg tgcaattcat gccaaacgag ttacactcat gaaaaaggat 420
ttggagctcg cgcgaaggat tgggggcaga gagaggggat gg 462
<210> 97
<211> 154
<212> PRT
<213> Beta vulgaris
<400> 97
Met Arg Val Lys His Thr Ala Ala Arg Lys Ser Thr Thr Asn Gly Pro
1 5 10 15
Arg Ser Lys Ala Gln Lys Ser Pro Arg Ser Leu Gln Ser Pro Gln Ser
20 25 30
Pro Ser Ser Ser Ser Lys Arg Lys Ser Arg Arg Asn Thr Asp Ala Thr
35 40 45
Pro Gln Lys Lys Lys Ala Tyr Arg Arg Lys Pro Gly Thr Val Ala Leu
50 55 60
Trp Glu Ile Arg Lys Phe Gln Lys Ser Phe Lys Pro Leu Ile Pro Ala
65 70 75 80
Ala Pro Phe Ile Arg Thr Val Arg Glu Ile Thr His Gln Phe Ala Pro
85 90 95
Tyr Val Gly Arg Trp Gln Ala Glu Ala Leu Met Ala Leu Leu Glu Ala
100 105 110
Ala Glu Asn Phe Ile Val Arg Leu Phe Glu Asp Gly Met Leu Cys Ala
115 120 125
Ile His Ala Lys Arg Val Thr Leu Met Lys Lys Asp Leu Glu Leu Ala
130 135 140
Arg Arg Ile Gly Gly Arg Glu Arg Gly Trp
145 150
Claims (19)
1.植物,具有单倍体诱导物生物活性并且包含编码包含CATD结构域的着丝粒组蛋白H3(CENH3)蛋白质的核苷酸序列,其中所述核苷酸序列包含在CATD结构域中导致CENH3蛋白质的氨基酸序列改变的突变,所述改变赋予单倍体诱导物生物活性。
2.权利要求1的植物,其中所述突变在SEQ ID No.38所示的来源于拟南芥(Arabidopsis thaliana)的CENH3蛋白质的环1中导致赋予单倍体诱导物生物活性的改变,所述环1对应于SEQ ID No.37的位置340-378的核苷酸并且位于CATD结构域内,或者所述突变在SEQ ID No.38所示的来源于拟南芥的CENH3蛋白质的α2-螺旋中导致赋予单倍体诱导物生物活性的改变,所述α2-螺旋对应于SEQ ID No.37的位置379-465的核苷酸并且位于CATD结构域内。
3.权利要求1或2的植物,其中所述突变导致表1或2所示的确定的氨基酸的取代或缺失。
4.前述权利要求任一项的植物,其中所述突变导致SEQ ID No.1或49的确定的氨基酸的取代或缺失。
5.前述权利要求任一项的植物,其中所述植物与野生型植物或者表达野生型CENH3蛋白质的植物之间的杂交产生至少0.1%的单倍体后代。
6.前述权利要求任一项的植物,其中包含所述突变的核苷酸序列是内源基因或者转基因。
7.权利要求6的植物,其中包含所述突变的核苷酸序列是转基因,并且编码CENH3蛋白质的至少一个内源基因被失活或者敲除。
8.前述权利要求任一项的植物,其中在SEQ ID No.49的位置2的氨基酸天冬酰胺被取代,优选取代为缬氨酸,或者在SEQ ID No.55的位置95的氨基酸丙氨酸被取代,优选取代为缬氨酸,或者在SEQ ID No.49的位置6的氨基酸脯氨酸被取代,优选取代为丝氨酸,或者在SEQ ID No.52的位置121的氨基酸脯氨酸被取代,优选取代为丝氨酸,或者在SEQ ID No.49的位置12的氨基酸色氨酸被取代,优选取代为终止信号,或者在SEQ ID No.52的位置127的氨基酸色氨酸被取代,优选取代为终止信号,或者在SEQ ID No.1的位置1的氨基酸丙氨酸被取代,优选取代为苏氨酸,或者在SEQ ID No.58的位置107的氨基酸丙氨酸被取代,优选取代为苏氨酸,或者在SEQ ID No.1的位置4的氨基酸亮氨酸被取代,优选取代为苯丙氨酸、异亮氨酸或者谷氨酰胺,或者在SEQ ID No.52的位置132或者在SEQ ID No.34的位置92或者在SEQ ID No.38的位置130或者在SEQ ID No.61的位置106的氨基酸亮氨酸被取代,优选取代为苯丙氨酸、异亮氨酸或者谷氨酰胺,或者在SEQ ID No.1的位置7的氨基酸亮氨酸被取代,优选取代为脯氨酸,或者在SEQ ID No.61的位置109的氨基酸亮氨酸被取代,优选取代为脯氨酸,或者在SEQ ID No.1的位置8的氨基酸谷氨酰胺被取代,优选取代为终止信号或亮氨酸,或者在SEQ ID No.58的位置114或者在SEQ ID No.61的位置110的氨基酸谷氨酰胺被取代,优选取代为终止信号或亮氨酸,或者在SEQ ID No.1的位置10的氨基酸丙氨酸被取代,优选取代为苏氨酸,或者在SEQ ID No.52的位置138的氨基酸丙氨酸被取代,优选取代为苏氨酸,或者在SEQ ID No.1的位置25的氨基酸半胱氨酸被取代,优选取代为酪氨酸,或者在SEQ ID No.52的位置153的氨基酸半胱氨酸被取代,优选取代为酪氨酸,或者在SEQID No.1的位置26的氨基酸丙氨酸被取代,优选取代为缬氨酸,或者在SEQ ID No.52的位置154的氨基酸丙氨酸被取代,优选取代为缬氨酸。
9.前述权利要求任一项的植物的部分,其优选是芽营养器官、根、花或花器官、种子、果实、胚珠、胚、植物组织或细胞。
10.单倍体植物,可通过权利要求1-8任一项的植物与表达野生型CENH3蛋白质的植物杂交获得。
11.双单倍体植物,可通过将权利要求10的单倍体植物转变为双单倍体植物获得,优选通过秋水仙素处理获得。
12.一种产生单倍体植物的方法,包括如下步骤:
a)将权利要求1-8的植物与表达野生型CENH3蛋白质的植物杂交,及
b)鉴别从杂交步骤产生的单倍体后代植物。
13.一种产生双单倍体植物的方法,包括如下步骤:
a)将权利要求1-8的植物与表达野生型CENH3蛋白质的植物杂交,
b)鉴别从杂交步骤中产生的单倍体后代植物,及
c)将所述单倍体后代植物转变为双单倍体植物,优选通过秋水仙素处理或者通过染色体自然加倍法转变。
14.促进细胞质交换的方法,包括如下步骤:
x)将作为胚珠亲本的权利要求1-8的植物与作为花粉亲本的表达野生型CENH3蛋白质的植物杂交,及
y)获得包含所述花粉亲本染色体和胚珠亲本细胞质的单倍体后代植物。
15.一种产生权利要求1-8的植物的方法,包括如下步骤:
i)对植物种子进行足量诱变剂甲基磺酸乙酯处理以获得M1植物,
ii)充分产生可育的M2植物,
iii)分离M2植物的基因组DNA,及
iv)选择在CENH3的CATD结构域中具有氨基酸序列改变的个体。
16.核苷酸序列,编码CENH3蛋白质的至少CATD结构域,包含在CATD结构域中导致CENH3蛋白质的氨基酸序列改变的突变。
17.载体,包含权利要求16的核苷酸序列。
18.植物细胞或宿主细胞,包含权利要求16的核苷酸序列或者权利要求17的载体作为转基因。
19.一种产生权利要求1-8的植物的方法,包括如下步骤:
yy)用权利要求16的核苷酸序列或者权利要求17的载体转化植物细胞,及
zz)从所述植物细胞再生具有单倍体诱导物生物活性的植物。
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