CN110042108A - 一种水稻株型生长发育相关编码基因及其应用 - Google Patents
一种水稻株型生长发育相关编码基因及其应用 Download PDFInfo
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- CN110042108A CN110042108A CN201910251175.2A CN201910251175A CN110042108A CN 110042108 A CN110042108 A CN 110042108A CN 201910251175 A CN201910251175 A CN 201910251175A CN 110042108 A CN110042108 A CN 110042108A
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Abstract
本发明公开了一种水稻株型生长发育相关编码基因,所述水稻株型生长发育相关编码基因名称为OrMKK3‑2,所述水稻株型生长发育相关编码基因OrMKK3‑2在调控水稻株型生长发育中的应用。本发明首次鉴定了与水稻株型生长发育相关基因OrMKK3‑2,水稻株型生长发育相关基因OrMKK3‑2在功能增强或表达量上升的条件下会引起水稻株型矮化、分蘖增多证明水稻株型生长发育相关基因蛋白或其蛋白在控制水稻株型中发挥重要作用。
Description
技术领域
本发明涉及基因工程技术领域,特别涉及一种水稻株型生长发育相关编码基因及其应用。
背景技术
水稻是人类最重要的粮食作物之一,世界上以稻米为主食的人口约占50%。亚洲地区对稻米的需求量逐年增加,由于经济快速发展、人口压力增大、工业用地剧增、耕地面积减少,水稻生产面临巨大的压力。因此持续提高和挖掘水稻的高产潜力,是科学家们的主要研究方向。水稻株型与品质、产量有着密切的关系。水稻株型在群体受光姿态、光能、水肥、空间利用率、产量提高和品质提升上发挥关键作用。株型主要取决于株型、叶型、分蘖数目、分蘖角度和穗部形态等几个方面。近年来,使用分子生物学方法克隆水稻株型基因成为研究的热点,克隆的水稻株型基因对水稻矮化育种、株型改良有着重要的理论和现实指导意义。
目前已经克隆明确功能的水稻株型基因达到21个,包括水稻1号染色体的sd1,d2,d10,d18,d61,gid2,gid1等基因控制着半矮秆和矮杆性状,2号染色体的gid2基因控制矮杆性状,3号染色体d14,brd1,fc1,OsDWARF4基因控制株型形成,4号染色体的d11基因控制水稻多蘖矮杆,5号染色体的eui1控制穗颈节间伸长,6号染色体d3和d35控制矮杆性状,7号染色体d6基因,8号染色体SPY,IPA1基因,10号染色体的brd2基因均控制水稻株型的形成。然而目前已发掘并鉴定的控制水稻株型性状的基因个体综合农艺性状表现较差,当今株型育种的主要问题是可应用的株型基因太少,如何突破瓶颈应用新的株型基因显得尤为关键。药用野生稻中蕴藏着丰富的基因,挖掘药用野生稻的有利基因。从野生稻中发掘并鉴定株型新资源,深入探究调控株型的分子机制,将有助于水稻育种家在育种过程中为分子设计育种改良水稻株叶型提供理论依据,最终实现高效育种。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
本发明针对上述技术问题,发明一种水稻株型生长发育相关编码基因及其应用。
为实现上述目的,本发明提供的技术方案如下:
一种水稻株型生长发育相关编码基因,名称为OrMKK3-2,来源于稻属药用野生稻(Oryza officinalis Wall.ex wstt)的Y003株系的cDNA序列,属于同一个基因的4种可变剪切开放阅读框的第二种开放阅读框。
其中,所述水稻株型生长发育相关编码基因OrMKK3-2为a)或b):
a)cDNA序列如SEQ ID No.1所示的基因序列,由1556个碱基组成;
b)将SEQ ID No.1所示的基因序列经过一个或几个碱基的取代和/或缺失和/或添加得到的具有水稻株型相关基因编码的OrMKK3-2蛋白活性的由a)衍生的蛋白质。
一种水稻株型生长发育相关编码基因,名称为OrMKK3-2,在调控水稻株型及分蘖发育中的应用。
其中,所述的调控水稻株型生长发育是指调控水稻矮化、多分蘖。
为解决上述技术问题,本发明还提供了所述水稻株型生长发育相关基因OrMKK3-2在培育矮秆、多分蘖水稻的转基因水稻中的应用。
其中,与所述水稻株型生长发育相关基因OrMKK3-2相关的生物材料,在培育矮秆、多分蘖水稻的转基因水稻中的应用;所述与水稻株型生长发育相关基因OrMKK3-2相关的生物材料为下述A1)至A20)中的任一种:
A1)核酸分子1;所述核酸分子1为编码所述水稻株型生长发育相关基因OrMKK3-2的核酸分子;
A2)含有A1)所述核酸分子1的表达盒;
A3)含有A1)所述核酸分子1的重组载体;
A4)含有A2)所述表达盒的重组载体;
A5)含有A1)所述核酸分子1的重组微生物;
A6)含有A2)所述表达盒的重组微生物;
A7)含有A3)所述重组载体的重组微生物;
A8)含有A4)所述重组载体的重组微生物;
A9)含有A1)所述核酸分子1的转基因植物细胞系;
A10)含有A2)所述表达盒的转基因植物细胞系;
A11)含有A3)所述重组载体的转基因植物细胞系;
A12)含有A4)所述重组载体的转基因植物细胞系;
A13)含有A1)所述核酸分子1的转基因植物组织;
A14)含有A2)所述表达盒的转基因植物组织;
A15)含有A3)所述重组载体的转基因植物组织;
A16)含有A4)所述重组载体的转基因植物组织;
A17)含有A1)所述核酸分子1的转基因植物器官;
A18)含有A2)所述表达盒的转基因植物器官;
A19)含有A3)所述重组载体的转基因植物器官;
A20)含有A4)所述重组载体的转基因植物器官。
上述与所述水稻株型生长发育相关基因OrMKK3-2相关的生物材料在调控水稻株型生长发育中的应用或在培育矮秆水稻的转基因水稻中的应用中,所述核酸分子可以是DNA,如cDNA、基因组DNA或重组DNA;所述核酸分子也可以是RNA,如mRNA或hnRNA等。
上述与所述水稻株型生长发育相关基因OrMKK3-2相关的生物材料中,所述表达盒是指能够在宿主细胞中表达相应蛋白质的DNA,该DNA不但可包括启动相关基因转录的启动子,还可包括终止相关基因转录的终止子,如A2)所述的含有编码所述水稻株型生长发育相关蛋白OrMKK3-2的核酸分子的表达盒,是指能够在宿主细胞中表达所述水稻株型生长发育相关基因OrMKK3-2的DNA。
可用现有的植物表达载体构建含有所述OrMKK3-2基因表达盒的重组载体,如pET-28a、pCAMBIA2301、pSP72、pROKII、pBin438、pCAMBIA1302、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa或pCAMBIA1391-Xb(CAMBIA公司)等。
上述生物材料中,A5)-A8)中任一所述重组微生物或B5)-B8)中任一所述重组微生物具体可为细菌、酵母、藻或真菌。其中,细菌可来自埃希氏菌属(Escherichia)、欧文氏菌(Erwinia)、根癌农杆菌属(Agrobacterium)、黄杆菌属(Flavobacterium)、产碱菌属(Alcaligenes)、假单胞菌属(Pseudomonas)、芽胞杆菌属(Bacillus)等。A9)-A12)中任一所述的转基因细胞系,A13)-A16)中任一所述的转基因植物组织,A17)-A20)中任一所述的转基因植物器官不包括植物的繁殖材料。
本发明所提供的一种培育水稻矮化、多分蘖的转基因水稻的方法,将SEQ ID No.1所示的水稻株型生长发育相关编码基因OrMKK3-2构建过表达载体导入水稻中,获得OrMKK3-2基因表达水平提高的转基因水稻:是过表达受体水稻中所述水稻株型生长发育相关基因OrMKK3-2的编码基因的表达,得到矮化的转基因水稻。
上述方法中,所述水稻株型生长发育相关基因OrMKK3-2的编码基因的表达是通过将如SEQ ID No.1的DNA片段导入受体水稻中实现的。
上述方法中,所述水稻株型生长发育相关基因OrMKK3-2的编码基因的表达可通过过表达载体导入到受体水稻中实现的。
其中,所述过表达载体为重组表达载体PMDC32或载体pCAMBIA1301;所述重组表达载体PMDC32是将表达载体PMDC32或载体pCAMBIA1301的Asc I和PacI识别位点间的序列替换为SEQ ID No.1所示的DNA序列。
其中,所述重组表达载体PMDC32可通过使用农杆菌介导、Ti质粒,植物病毒载体,直接DNA转化,微注射,电穿孔等常规生物技术方法导入植物细胞或组织。
其中,所述方法还包括从导入SEQ ID No.1所示的DNA分子的受体水稻中筛选所述水稻株型生长发育相关基因OrMKK3-2表达量升高的水稻,得到所述OrMKK3-2基因表达水平升高的转基因水稻的步骤。
其中,所述转基因水稻理解为不仅包含将所述基因转化受体水稻得到的第一代转基因水稻,也包括其子代。对于转基因水稻,可以在该物种中繁殖该基因,也可用常规育种技术将该基因转移进入相同物种的其它品种,特别包括商业品种中。所述转基因水稻包括种子、愈伤组织、完整植株和细胞。
上文中,所述水稻具体可为日本晴。
与现有技术相比,本发明具有如下有益效果:
本发明首次鉴定了与水稻株型生长发育相关基因OrMKK3-2,水稻株型生长发育相关基因OrMKK3-2在功能增强或表达量上升的条件下会引起水稻株型矮化、分蘖增多证明水稻株型生长发育相关基因蛋白或其蛋白在控制水稻株型中发挥重要作用。本发明不仅为进一步阐明水稻株型的分子机理提供基础,而且为水稻育种提供新的基因资源和育种资源。本发明获得的OrMKK3-2基因表达升高的转基因水稻,作为新的水稻种质材料,可用于研究水稻矮化、分蘖增多的分子机理和发现更多的调控水稻株型发育的基因。本发明对于通过遗传育种和基因工程方法利用该基因资源有效地调控水稻株型具有重要的应用价值。
附图说明
图1为OrMKK3-2基因表达水平升高的过表达转基因水稻的OrMKK3-2基因的转录水平的检测;其中,WT代表受体亲本日本晴植株,OE-OrMKK3-2-1代表转入重组载体PMDC32-OrMKK3-2的过表达阳性植株;OE-OrMKK3-2植株,OE-OrMKK3-2-2代表转入重组载体PMDC32-OrMKK3-2的过表达阳性植株OE-OrMKK3-2植株,OE-OrMKK3-2-1与OE-OrMKK3-2-2为不同的转化事件。
图2为OrMKK3-2基因表达水平升高的过表达转基因水稻的表型观察;其中,WT代表受体亲本日本晴植株,OE-OrMKK3-2-1代表转入重组载体PMDC32-OrMKK3-2的过表达阳性植株OE-OrMKK3-2植株,OE-OrMKK3-2-2代表转入重组载体PMDC32-OrMKK3-2的过表达阳性植株OE-OrMKK3-2植株,OE-OrMKK3-2-1与OE-OrMKK3-2-2为不同的转化事件。
具体实施方式
下面结合附图具体实施方式进行详细描述,但应当理解本发明的保护范围并不受具体实施方式的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的水稻日本晴(也称为野生型水稻,简称WT),为市售所得,公众也可从广西壮族自治区农业科学院水稻研究所获得该生物材料,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
下述实施例中所用表达载体PMDC32为市售所得,公众也可从中广西壮族自治区农业科学院水稻研究所获得该生物材料,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
下述实施例中的农杆菌为根癌农杆菌EHA105(Agrobacterium tumefaciensEHA105)(New Agrobacterium helper plasmids for gene transfer toplants.Hood,ElizabethE;Gelvin,StantonB;Melchers,Leo S;Hoekema,Andre.Transgenic research,2(4):p.208-218(1993)),为市售所得,公众也可从广西壮族自治区农业科学院水稻研究所获得该生物材料,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
实施例1
水稻株型生长发育相关基因OrMKK3-2基因过表达载体的构建
1、水稻株型生长发育相关基因OrMKK3-2基因的获得
以药用野生稻Y003(Oryza officinalis Wall.ex wstt)的反转录cDNA为模板,用如下引物primer1和primer2进行PCR扩增获得目的基因。
primer1:5'TTGGCGCGCCCTGCAGGTTTAAACGAATTGCCCTT 3';
primer2:5'CCTTAATTAATGCTGCTCTCCCTTCCTTTTGTGCT 3'。
将PCR产物回收纯化后连接入Zero(购买自北京全式金公司)测序载体,转化DH5α感受态细胞,挑选阳性克隆后,进行测序。
测序结果表明,扩增得到的PCR产物有4个可变剪切,第二个为序列如SEQ ID No.1所示的核苷酸序列,长度为1556bp,命名为OrMKK3-2基因。OrMKK3-2基因编码的蛋白质的氨基酸序列如SEQ ID No.2所示。
2、水稻株型生长发育相关基因OrMKK3-2基因过表达载体(重组表达载体PMDC32-OrMKK3-2)的构建
1)用primer1和primer2扩增药用野生稻cDNA,获得OrMKK3-2基因的可变剪切,并连接到重组载体Zero-OrMKK3-2,得到了Zero-OrMKK3-2的阳性克隆。用限制性内切酶Asc I和PacI酶切重组载体Zero-OrMKK3-2获得OrMKK3-2片段;
2)用限制性内切酶Asc I和PacI酶切表达载体PMDC32,得到线性表达载体PMDC32,回收该线性片段;将步骤1)中得到的片段OrMKK3-2采用同源重组定向克隆的方法整合到线性表达载体PMDC32上(具体方法参考PMDC32说明书,),得到同源重组产物1,再将同源重组产物1转入DH5α感受态细胞,37℃培养过夜,得到重组载体PMDC32-OrMKK3-2;
3)对步骤2)所得重组载体PMDC32-OrMKK3-2进行测序,结果表明该重组载体PMDC32-OrMKK3-2是在表达载体PMDC32的Asc I酶切位点正向***了如SEQ ID No.1所示的核苷酸序列,即成功将PMDC32的Asc I和PacI识别位点(识别序列)间的DNA序列替换为如SEQ ID No.1所示的DNA序列。
实施例2
培育水稻株型生长发育相关基因OrMKK3-2基因表达水平升高的过表达OrMKK3-2转基因植株及转基因植株的鉴定
一、培育OrMKK3-2基因表达水平升高的过表达OrMKK3-2转基因植株将重组载体PMDC32-OrMKK3-2通过根癌农杆菌EHA105介导转化日本晴粳稻,具体方法如下:
1、将实施例1所得重组载体PMDC32-OrMKK3-2用热激法导入根癌农杆菌EHA105中得到含有重组载体PMDC32-OrMKK3-2的重组根癌农杆菌EHA105;将含有重组载体PMDC32-OrMKK3-2的重组根癌农杆菌EHA105在28℃培养16h,收集菌体;采用含有浓度为100μM乙酰丁香酮的N6液体培养基(Sigma,产品目录号为C1416)将菌体进行稀释,得到稀释菌液,稀释菌液的OD600≈0.5;
2、将培养至一个月的水稻成熟胚胚性愈伤组织与步骤1的稀释菌液混合侵染30min,采用滤纸吸干菌液后转入N6固体共培养培养基中,在24℃共培养3d,得到共培养处理后的愈伤组织;
3、将步骤2的共培养处理后的愈伤组织接种在含有质量浓度为150mg/L潮霉素的N6固体筛选培养基(向N6固体培养基中加入潮霉素得到N6固体筛选培养基,N6固体筛选培养基中潮霉素的质量浓度为150mg/L)上进行第一次筛选;
4、在第一次筛选开始的第16天挑取健康愈伤组织转入含有质量浓度为200mg/L潮霉素的N6固体筛选培养基(向N6固体培养基中加入潮霉素得到N6固体筛选培养基,N6固体筛选培养基中潮霉素的质量浓度为200mg/L)上进行第二次筛选,每15天继代一次,共继代1次;
5、挑取步骤4获得的抗性愈伤组织转入含有质量浓度为150mg/L潮霉素的分化培养基上(分化培养基:6-BA 2mg,NAA 0.2mg,N6 4g,水解酪蛋白1g,肌醇0.1g,蔗糖25g,山梨醇2.4g,琼脂粉7g,去离子水1L)进行分化,在24℃培养45d(此时植株地上部分高度约为15cm),打开瓶口炼苗3天,然后移栽至温室栽培,即为转PMDC32-OrMKK3-2植株(T0代)。为不同的转化事件命名为OE-OrMKK3-2-1与OE-OrMKK3-2-2分别代表转入重组载体PMDC32-OrMKK3-2的过表达阳性植株。
二、水稻株型生长发育相关基因OrMKK3-2基因表达水平升高的PMDC32-OrMKK3-2转基因植株的PCR鉴定提取
步骤一获得的转PMDC32-OrMKK3-2(OE-OrMKK3-2-1、OE-OrMKK3-2-2)植株的T0代幼苗和受体亲本水稻日本晴植株的幼苗(简称为WT)的基因组DNA,并采用引物primer1:5'TTGGCGCGCCCTGCAGGTTTAAACGAATTGCCCTT 3'和primer2:5'CCTTAATTAATGCTGCTCTCCCTTCCTTTTGTGCT 3',进行PCR分子检测鉴定阳性苗,得到1556bp PCR产物的植株为阳性苗,即以上提到的OE-OrMKK3-2-1、OE-OrMKK3-2-2。
三、水稻株型生长发育相关基因OrMKK3-2基因表达水平升高的过表达转基因植株的OrMKK3-2基因表达水平的鉴定:
分别提取步骤二得到的OE-OrMKK3-2-1、OE-OrMKK3-2-2植株和受体亲本水稻日本晴植株(简称为WT)叶片的RNA,设定内参为Actin,利用内参引物Actin-F和Actin-R,以及OrMKK3-2基因特异性定量引物OrMKK3-2-qRT-F和OrMKK3-2-qRT-R进行荧光定量PCR反应来检测不同转基因植株OrMKK3-2基因的表达水平的变化。结果表明(图1),在转入重组载体PMDC32-OrMKK3-2的阳性植株中OrMKK3-2基因的表达水平均比对照(WT)的OrMKK3-2基因的表达水平的显著上升,上述引物如下:
Actin-F:5’-ATTTGGCACCACACATTCTAC-3’
Actin-R:5’-ATAACCTTCGTAGATTGGGACT-3’;
OrMKK3-2-qRT-F:5'CCCCGCCTACTTCTTCTTTC 3'
OrMKK3-2-qRT-R:5'CGCCGCCTTATCCATCTC 3'。
四、水稻株型生长发育相关基因OrMKK3-2基因表达水平上升的OrMKK3-2过表达转基因植株的表型鉴定
分别将步骤二得到的OE-OrMKK3-2-1、OE-OrMKK3-2-2(同上)植株和受体亲本水稻日本晴植株(简称为WT)种植在海南实验基地,观察整个生长期内PMDC32-OrMKK3-2植株和受体亲本水稻日本晴植株(简称为WT)的表型差异。测量观察结果如图2、表1,与受体亲本水稻日本晴植株相比,PMDC32-OrMKK3-2植株均出现了株型矮化的表型,从而证明了OrMKK3-2基因参与控制水稻株型的生长发育,即该OrMKK3-2基因为水稻株型相关基因。
表1.水稻株型生长发育相关基因OrMKK3-2过表达转基因株高与分蘖表现
编号 | 株高(cm) | 株高标准误 | 分蘖(支) | 分蘖标准误 |
WT | 61.08 | 0.46 | 6.25 | 0.46 |
OE-OrMKK3-2-1 | 38.67 | 0.24 | 9.75 | 0.72 |
OE-OrMKK3-2-2 | 61.08 | 0.61 | 12.5 | 0.68 |
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
序列表
<110> 广西壮族自治区农业科学院水稻研究所
<120> 一种水稻株型生长发育相关编码基因及其应用
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atggcggggc tcgaggagct gaagaagaag ctgcagcccc tgctgttcga cgacccggac 60
aagggcggcg tcagtagcag ggttcctcta ccggaggaca cctgcgactc ctacgtggtt 120
tctgatggtg gaactgtgaa tttgttgagt agatcattgg gtgagtataa catcaatgag 180
catggctttc ataaacgaag cactgggcca gaggagtcag attctggtga aaaggcatac 240
cgatgtgcct ctcatgatat gcacatattt ggccccatcg gtaatggtgc aagcagtgtt 300
gtgcagagag ctgtttttat accagttcat cgaattttgg ccttgaagaa gataaatata 360
tttgagaagg agaagagaca acaaattctg aatgagatga gaacattatg tgaagcatgt 420
tgttatattg gtttagttga attccagggt gcattctaca tgcctgattc tggacaaata 480
agcatcgccc ttgaatacat ggatggtggg tccttagctg atgttataaa gattaagaaa 540
tcaataccag aaccagttct tgcacatatg ctgcagaaag tattgcttgg tttgcgctac 600
ttgcatgaag taagacatct agtgcataga gatataaagc cagcgaattt actggtaaat 660
ctcaagggcg aggcaaagat aacagatttt ggagtgagtg ctggtttgga taatacgatg 720
gccatggcac agtcacatat atgtcacctg agagaattcg taacgagaat tactcttatg 780
ctgctgatat ttggagtctt ggactagcag tattggagtg tgctactggt aaatttccat 840
ataatgtgaa tgaaggccca gccaacctca tgctgcagat tctggatgat ccatcaccaa 900
caccaccaaa agatgcttat tcatccgagt ttagttcgtt catcaatgac tgcttgcaga 960
aagatgctga tgcaaggcct tcgtgtgagc agcttttgtc acacccattc atcaagaggt 1020
atgaaaacac taccatggac ttggtagctt acatcaaaag tgttgttgat ccaacagaaa 1080
gattaaagca aatagcagag atgcttgccg tacattacta cctcctcttt aacggcactg 1140
atgggatttg gcattatatg aagacattct acatggaaga atcaactttc agtttctcag 1200
ggaatgtgta tgttggccaa agtgacatat ttgatacttt atcaaatata agaaagaagt 1260
taacaggtga tcgtcctcga gagaaaattg ttcatgttgt tgagaagcta cattgtcgtg 1320
cacacggaga aacaggaata gctatacgtg tgtctggatc gttcattgtg ggaaaccaat 1380
ttctaatatg tggtgaaggg ttgcaagctg aaggaatgcc aagcttggag gaactctcca 1440
ttgatattcc aagcaagcgg gtaggtcagt tccgggagca atttatcatg gagccaggaa 1500
gttccatggg atgctactac atattaaggc aagatctata catcatccaa gcctga 1556
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Met Ala Gly Leu Glu Glu Leu Lys Lys Lys Leu Gln Pro Leu Leu Phe
1 5 10 15
Asp Asp Pro Asp Lys Gly Gly Val Ser Ser Arg Val Pro Leu Pro Glu
20 25 30
Asp Thr Cys Asp Ser Tyr Val Val Ser Asp Gly Gly Thr Val Asn Leu
35 40 45
Leu Ser Arg Ser Leu Gly Glu Tyr Asn Ile Asn Glu His Gly Phe His
50 55 60
Lys Arg Ser Thr Gly Pro Glu Glu Ser Asp Ser Gly Glu Lys Ala Tyr
65 70 75 80
Arg Cys Ala Ser His Asp Met His Ile Phe Gly Pro Ile Gly Asn Gly
85 90 95
Ala Ser Ser Val Val Gln Arg Ala Val Phe Ile Pro Val His Arg Ile
100 105 110
Leu Ala Leu Lys Lys Ile Asn Ile Phe Glu Lys Glu Lys Arg Gln Gln
115 120 125
Ile Leu Asn Glu Met Arg Thr Leu Cys Glu Ala Cys Cys Tyr Ile Gly
130 135 140
Leu Val Glu Phe Gln Gly Ala Phe Tyr Met Pro Asp Ser Gly Gln Ile
145 150 155 160
Ser Ile Ala Leu Glu Tyr Met Asp Gly Gly Ser Leu Ala Asp Val Ile
165 170 175
Lys Ile Lys Lys Ser Ile Pro Glu Pro Val Leu Ala His Met Leu Gln
180 185 190
Lys Val Leu Leu Gly Leu Arg Tyr Leu His Glu Val Arg His Leu Val
195 200 205
His Arg Asp Ile Lys Pro Ala Asn Leu Leu Val Asn Leu Lys Gly Glu
210 215 220
Ala Lys Ile Thr Asp Phe Gly Val Ser Ala Gly Leu Asp Asn Thr Met
225 230 235 240
Ala Met Ala Gln Ser His Ile Cys His Leu Arg Glu Phe Val Thr Arg
245 250 255
Ile Thr Leu Met Leu Leu Ile Phe Gly Val Leu Asp Gln Tyr Trp Ser
260 265 270
Val Leu Leu Val Asn Phe His Ile Met Met Lys Ala Gln Pro Thr Ser
275 280 285
Cys Cys Arg Phe Trp Met Ile His His Gln His His Gln Lys Met Leu
290 295 300
Ile His Pro Ser Leu Val Arg Ser Ser Met Thr Ala Cys Arg Lys Met
305 310 315 320
Leu Met Gln Gly Leu Arg Val Ser Ser Phe Cys His Thr His Ser Ser
325 330 335
Arg Gly Met Lys Thr Leu Pro Trp Thr Trp Leu Thr Ser Lys Val Leu
340 345 350
Leu Ile Gln Gln Lys Asp Ser Lys Gln Arg Cys Leu Pro Tyr Ile Thr
355 360 365
Thr Ser Ser Leu Thr Ala Leu Met Gly Phe Gly Ile Ile Arg His Ser
370 375 380
Thr Trp Lys Asn Gln Leu Ser Val Ser Gln Gly Met Cys Met Leu Ala
385 390 395 400
Lys Val Thr Tyr Leu Ile Leu Tyr Gln Ile Glu Arg Ser Gln Val Ile
405 410 415
Val Leu Glu Arg Lys Leu Phe Met Leu Leu Arg Ser Tyr Ile Val Val
420 425 430
His Thr Glu Lys Gln Glu Leu Tyr Val Cys Leu Asp Arg Ser Leu Trp
435 440 445
Glu Thr Asn Phe Tyr Val Val Lys Gly Cys Lys Leu Lys Glu Cys Gln
450 455 460
Ala Trp Arg Asn Ser Pro Leu Ile Phe Gln Ala Ser Gly Val Ser Ser
465 470 475 480
Gly Ser Asn Leu Ser Trp Ser Gln Glu Val Pro Trp Asp Ala Thr Thr
485 490 495
Tyr Gly Lys Ile Tyr Thr Ser Ser Lys Pro
500 505
Claims (9)
1.一种水稻株型生长发育相关编码基因,其特征在于:所述水稻株型生长发育相关编码基因名称为OrMKK3-2;
其中,所述水稻株型生长发育相关编码基因OrMKK3-2为a)或b):
a)cDNA序列如SEQ ID No.1所示的基因序列,由1556个碱基组成;
b)将SEQ ID No.1所示的基因序列经过一个或几个碱基的取代和/或缺失和/或添加得到的具有水稻株型相关基因编码的OrMKK3-2蛋白活性的由a)衍生的蛋白质。
2.根据权利要求1所述水稻株型生长发育相关编码基因,其特征在于:在调控水稻株型及分蘖发育中的应用。
3.根据权利要求2所述水稻株型生长发育相关编码基因,其特征在于:所述的调控水稻株型生长发育是指调控水稻矮化、多分蘖。
4.根据权利要求2或3所述水稻株型生长发育相关编码基因,其特征在于:所述水稻株型生长发育相关基因OrMKK3-2在培育矮秆、多分蘖水稻的转基因水稻中的应用。
5.一种利用如权利要求1所述基因培育矮化、多分蘖的转基因水稻的方法,其特征在于:将SEQ ID No.1所示的水稻株型生长发育相关编码基因OrMKK3-2构建过表达载体导入水稻中,获得OrMKK3-2基因表达水平提高的转基因水稻:是过表达受体水稻中所述水稻株型生长发育相关基因OrMKK3-2的编码基因的表达,得到矮化的转基因水稻。
6.根据权利要求5所述的方法,其特征在于:所述过表达载体为重组表达载体PMDC32或载体pCAMBIA1301;所述重组表达载体PMDC32是将表达载体PMDC32或载体pCAMBIA1301的Asc I和PacI识别位点间的序列替换为SEQ ID No.1所示的DNA序列。
7.根据权利要求5所述的方法,其特征在于:所述重组表达载体PMDC32可通过使用农杆菌介导、Ti质粒,植物病毒载体,直接DNA转化,微注射,电穿孔等常规生物技术方法导入植物细胞或组织。
8.根据权利要求5所述的方法,其特征在于:所述方法还包括从导入SEQ ID No.1所示的DNA分子的受体水稻中筛选所述水稻株型生长发育相关基因OrMKK3-2表达量升高的水稻,得到所述OrMKK3-2基因表达水平升高的转基因水稻的步骤。
9.根据权利要求5所述的方法,其特征在于:所述水稻具体可为日本晴。
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