CN106967726B - 一种创建亚洲栽培稻与非洲栽培稻种间杂种亲和系的方法和应用 - Google Patents
一种创建亚洲栽培稻与非洲栽培稻种间杂种亲和系的方法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于基因编辑技术创建亚洲栽培稻与非洲栽培稻种间杂种亲和系的方法及其在水稻远缘杂种优势中的应用。即针对水稻种间杂种不育S1座位的非洲稻候选基因OgTPR1选取合适的基因编辑位点,以CRISPR/Cas9等定点编辑技术对OgTPR1进行定点敲除。在证明敲除突变基因ogtpr1不影响亲本花粉和雌配子育性的基础上,以ogtpr1突变系与亚洲栽培稻杂交的杂种F1的花粉和雌配子育性正常,即消除了种间杂种不育的遗传效应。本发明提供的技术方法能有效地打破S1座位介导的种间杂种生殖障碍,对利用水稻种间远缘杂种优势,提高粮食产量具有重要的应用价值。
Description
技术领域
本发明属于植物基因工程技术和作物遗传育种技术领域,具体涉及一种创建亚洲栽培稻与非洲栽培稻种间杂种亲和系的方法及其在水稻远缘杂种优势利用中的应用。
背景技术
全球面临人口增多而可耕种土地日益减少的矛盾,利用作物杂种优势,提高农作物产量对化解粮食危机有着重要的意义。两亲本亲缘关系较远缘,即两亲本间的遗传差异较大,杂种后代优势表现就更明显,称为杂种优势。然而,远缘杂种通常伴随较严重的杂种不育现象,这极大地阻碍了作物杂种优势的应用。杂种不育是生物种间或亚种杂种普遍存在的现象,是一种物种分化过程中产生的生殖隔离机制。目前,我国杂交水稻是提高水稻产量的重要途径。因此克隆作物杂种不育基因及阐明其分子机理,对打破种间或亚种生殖壁垒,充分利用杂种优势提高产量和抗性,对农业生产具有重大意义。
稻属(Oryza)属于禾本科,有22个种,包括20个野生稻种和2个栽培稻种。水稻生产中所用的栽培稻种,一个是起源于亚洲的亚洲栽培稻(Oryza sativa L.),另一个是起源于非洲的非洲栽培稻(Oryza glaberrima Steud)。亚洲栽培稻具有产量高和品质优等优良性状,而非洲栽培稻具有耐逆性强(耐高温,耐旱等)优良性状。水稻种间如亚洲栽培稻与非洲栽培稻之间的杂种优势比亚种间如籼稻(O. sativa. ssp. indica)与粳稻(O. sativa. ssp. japonica)之间的杂种优势表现更明显,但相应的杂种不育现象更为突出。水稻种间或亚种间杂种不育是由多个基因座位控制的。其中S1座位是控制亚洲栽培稻和非洲栽培稻种间杂种(简称亚非稻杂种)不育性的主要遗传因子。杂种中杂合的S1座位的等位基因遗传互作导致杂种的雄配子(花粉)和雌配子半不育,即含有亚洲栽培稻等位基因S1-s的花粉和雌配子败育,而含有非洲栽培稻等位基因S1-g的花粉和雌配子正常可育(Koide等, 2008,New Phytologist,179, 888-900.)。打破杂种不育的常规育种方法是通过筛选选育含有杂种不育座位的亲和型等位基因,以多代回交的方法培育杂种亲和系。但种间杂种不育由于物种亲缘关系较远,一般不存在相应的亲和型等位基因,这使得种间杂种不育难以被打破,因此目前尚未有克服水稻种间杂种不育的可行性技术方法。
基因编辑技术是近年来发展起来的高效的基因修饰改造技术,可以定点敲除多种生物的内源基因。相比较早期发展的辛指核酸酶基因编辑技术,近年来发展的TALLENs***、CRISPR/Cas9以及CRISPR/Cpf1***的效率更高,尤其是CRISPR/Cas9***的技术更加成熟。
马生健于2015年4月公开了一个亚非稻杂种不育座位S1的相关基因ORF12(等同于本发明所述的OgTPR1)的相关专利的申请(公开号:CN 104562 A)。该ORF12是仅基于基因定位分析,实际上是S1座位定位区(18 kb)内含有的7个候选基因之一。但该专利公开并没有验证ORF12在S1杂种不育的功能以及是否水稻花粉和雌配子发育所必需,也没有提供如何创建杂种亲和系,以克服种间杂交育种中的S1杂种不育的技术方法。
发明内容
本发明所要解决的技术问题是克服现有育种技术存在的缺陷,提供一种快速创建亚非稻种间杂种亲和系的方法。
本发明的第二个目的是提供所述方法在水稻远缘杂种优势利用中的应用。
本发明的目的是通过以下技术方案予以实现的:
亚洲栽培稻与非洲栽培稻种间杂种不育S1座位相关的OgTPR1基因在克服所述种间杂种不育中的应用,所述OgTPR1基因的编码序列如SEQ ID NO:1所示。
本发明还提供OgTPR1基因的定点敲除突变基因ogtpr1,所述定点敲除突变基因ogtpr1的序列如SEQ ID NO:2~3所示。
本发明利用一种基因定点编辑技术,优选的是CRISPR/Cas9基因编辑技术,对杂种不育S1座位相关的OgTPR1基因进行定点编辑以敲除其功能,证明OgTPR1的功能丧失突变不影响突变系的花粉和雌配子育性。以此突变系与亚洲栽培稻杂交产生的杂种的花粉和雌配子育性正常,即此突变系为本发明创建的S1座位亲和系,可以用于打破亚非稻杂种的生殖壁垒。
本发明还提供含有权利要求2所述定点敲除突变基因ogtpr1的转化体。
本发明还提供所述定点敲除突变基因ogtpr1或者所述转化体在克服亚洲栽培稻与非洲栽培稻种间杂种不育中的应用。
本发明还提供所述定点敲除突变基因ogtpr1或者所述转化体在创建亚洲栽培稻与非洲栽培稻种间杂种S1座位的亲和系中的应用。
本发明还提供一种亚洲栽培稻与非洲栽培稻种间杂种S1座位亲和系,是通过定点敲除OgTPR1,构建亚洲栽培稻与非洲栽培稻种间杂种S1座位亲和系,包括以下步骤:
S1. 针对OgTPR1基因的序列,以一种基因定点编辑***,选择OgTPR1中的1个或多个靶点,构建目的基因编辑转化载体或其表达的核酸酶蛋白和引导RNA;
S2. 将目的基因编辑转化载体或其表达的核酸酶蛋白和引导RNA导入含有OgTPR1的非洲稻受体材料,获得敲除OgTPR1基因功能的植株;
S3. 通过PCR扩增包含靶点的目的片段并进行测序分析突变情况,获得相应的OgTPR1基因功能敲除突变体ogtpr1,即获得与亚洲稻杂交的杂种S1座位亲和系。
优选地,S1所述的靶点序列如SEQ ID NO:4所示。
本发明还提供所述亚洲栽培稻与非洲栽培稻种间杂种S1座位亲和系在克服亚洲栽培稻与非洲栽培稻的杂种不育中的应用。
具体地,所述应用是将所述的S1座位亲和系与亚洲栽培稻杂交,获得杂种一代及其后代,这些杂种的花粉和雌配子育性得以恢复正常。
具体的操作是:将所述ogtpr1亲和系与亚洲栽培稻杂交,获得突变型杂种F1(mF1),该mF1的育性得以恢复正常,而且其自交后代(mF2)的S1座位基因型的分离比恢复为孟德尔自由分离比(1:2:1)。
与现有技术相比,本发明具有以下有益效果:
(1)发明针对亚非稻杂种不育S1座位的候选基因OgTPR1,利用一种基因定点编辑技术将非洲稻S1-g等位基因座的OgTPR1敲除其功能,获得ogtpr1突变体,发现其自身的育性正常,首次证实OgTPR1不是雄配子和雌配子正常发育必需的基因,可以作为亲本用于杂交育种;(2)利用ogtpr1突变体与亚洲栽培稻杂交获得的杂种不再产生雄配子和雌配子的败育,首次证明OgTPR1是野生型非洲稻与亚洲稻的S1杂种不育的关键基因;(3)本发明首次提供一种以基因定点编辑技术快速创建杂种亲和系的方法,所述亲和系可用于克服亚非稻杂种不育性,有利于利用水稻种间杂种优势和综合利用双亲本的有利性状。而且,本发明获得的ogtpr1突变体可以通过后代分离,将转基因元件排除。因此,本发明具有明显的创新性和有益的效果,为挖掘水稻远缘杂种优势,提高水稻产量和抗性,提供了新的分子育种技术手段。
附图说明
图1为OgTPR1的基因结构和定点敲除载体的构建;图1A为OgTPR1的基因结构(小黑框表示外显子)及CRISPR/Cas9***的编辑位点,*和虚框内的红色碱基表示OgTPR1的编辑位点;图1B为利用Ma等(2015,Molecular Plant, 8, 1274-1284)报道的CRISPR/Cas9基因编辑载体***和构建方法,将由水稻OsU3启动子驱动的sgRNA的表达盒克隆到pYLCRISPR/Cas9Pubi-H基因编辑双元载体(KO-OgTPR1),以酶切验证该载体,#表示ASCI酶切的sgRNA表达盒目的条带。
图2为对含有非洲栽培稻OgTPR1的近等基因系(NIL-g)进行基因敲除获得突变体ogtpr1的鉴定;图2A为ogtpr1突变体的转基因T0植株的潮霉素抗性基因HPT的鉴定,*表示其中用于测序分析靶点突变情况的2个独立ogtpr1突变体的转基因鉴定条带;图2B为将2个独立突变体 ogtpr1-1,ogtpr1-2的包含靶点的片段通过PCR扩增和测序获得的波峰图。reference为野生型参考序列。
图3为ogtpr1功能敲除突变体的转基因T1代的不含转基因个体的鉴定及表型观察;图3A为以PCR扩增抗潮霉素基因HPT筛选不含转基因个体(以*指示)的鉴定,图3B中FF(Full Fertility)表示花粉和小穗为全可育。花粉标尺为100 μm;穗标尺为5 cm。
图4为基因编辑突变体ogtpr1(即亚非稻杂种亲和系)与粳稻亲本(RP-s)杂交产生的突变型杂种一代(mF1)的花粉和小穗育性正常;图4A,B分别为RP-s与其近等基因系(NIL-g,含野生型OgTPR1)的杂种F1和ogtpr1-1,ogtpr1-2与RP-s的杂种mF1的花粉(上图)和小穗(下图)育性;SS(Semi Sterility)表示半不育,FF表示全可育;图4C为F2和mF2的S1座位基因型分离比;χ2(1:2:1)检测,表示F2产生S1-s型雌雄配子选择性败育导致严重偏态分离,而mF2不产生雌雄配子选择性败育(没有偏态分离);***表示P值小于0.001,呈现极显著差异;g/g,g/s,s/s分别表示S1座位的S1-g/S1-g纯合基因型,S1-g/S1-s杂合基因型,和S1-s/S1- s纯合基因型。
具体实施方式
下面将结合说明书附图和具体实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤、条件所作的修改或替换,均属于本发明的范围。例如,对OgTPR1基因(包括其启动子)可以设计SEQ ID NO:4以外的其它1个或多个编辑靶点,也可以利用不同的基因定点编辑***(如TALLEN或CRISPR/Cpf1***)进行有效的功能敲除,包括缺失整个OgTPR1基因(包括其启动子)序列,均可达到同等的目的。若无特别说明,实施例中所用的实验方法均为本领域技术人员所熟知的常规方法和技术,所使用的试剂或材料均为通过商业途径得到。
实施例1 OgTPR1功能敲除载体的构建
针对亚非稻杂种不育基因OgTPR1(SEQ ID NO:1所示)的编码序列,设计靶标序列(SEQ ID NO:4)。利用本发明人的实验室创建的CRISPR/Cas9基因编辑载体***和构建方法(Ma等2015,Molecular Plant, 8, 1274-1284),合成2条互补引物(U3-OgTPR1-F, OgTPR1-R)形成靶点双链接头(表1),连接到由水稻OsU3启动子驱动的sgRNA的表达盒中。将PCR扩增的sgRNA的表达盒片段克隆到基因编辑双元载体pYLCRISPR/Cas9Pubi-H(T-DNA区含有),产生KO-OgTPR1(图1B)。
实施例2 OgTPR1基因敲除系ogtpr1的鉴定
将实施例1的双元转化载体KO-OgTPR1通过农杆菌介导转化NIL-g获得T0转化体。以引物HptF/HptR(表1)进行PCR鉴定得到转基因阳性敲除突变体(图2A)。进一步地,通过PCR扩增2个独立转化植株的一段含有靶点的片段,测序分析OgTPR1基因的突变效果,获得突变体 ogtpr1-1和ogtpr1-2(图2B)。它们由于单碱基***产生移码和提前终止密码,即功能丧失的截断基因ogtpr1,如SEQ ID NO:2~3所示。
实施例3 ogtpr1功能敲除突变体的T1植株的不含转基因个体的鉴定及表型观察
对上述ogtpr1功能敲除突变体的转基因T1植株进行转基因(HPT基因)的PCR检测,分离出不含转基因的个体(图3A)。然后观察2个ogtpr1突变体的育性,发现这些突变体的花粉和小穗表现为全可育(图3B)。
实施例4 基因编辑ogtpr1突变体与亚洲栽培稻的杂种的育性表现
利用不含转基因的ogtpr1突变体与粳稻亲本(RP-s)进行杂交获得杂种mF1。对mF1的育性进行观察发现其花粉和小穗的育性都正常,而含有野生型OgTPR1的近等基因系(NIL-g)与RP-s的杂种F1的花粉和小穗都是半不育(图4A,4B)。进一步对mF1的自交产生的mF2株系进行检测S1基因型的分离比分析,结果表明其分离比恢复了自由分离比(图4C)。即ogtpr1突变体可以作为亚非杂种亲和系与亚洲稻进行杂交育种。
SEQUENCE LISTING
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gccttcaaga atgagtggat ctcaggtgac ctccttcgga actctaagca agatgtttgg 120
actggtctca gtgatgggct taaatcatat ttgtctaaaa gtgtcgcttc tattattttg 180
ttcaatggag acgaaatctt attttcatgc tcaggcatcg ctatggaaca ccagtttttt 240
acaaagtttt tgactaccgc aactctggtt agagctctta atgccacgac caaacaccat 300
gatgacttaa agattcaagt gcgccttgat ggcaccagac tgtatgatgg gtatatggct 360
gaatatgatt tggataatga cttttctgtt gtcgaagtct atagtgtccg tgatgttcag 420
gttggacctt tccaaagtgc acttgaaagt ctgccccatg gtgaggtgtt agctgtaggg 480
cgtgacacct ccggcgaaat aatggtcaag actgtggagt tgaatggcga ttcaagggta 540
tctgaggatg atagagatct tcattgtaaa atctcaaagc cttgggaagg tgggccgctt 600
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tttctaccat ggggcacaac tctcaatcat tacttgacat tcgtgcaaaa gaagactggc 720
cttgtacaat caaaaaaaat gaaggttcac aggcctgaag catccatagg tgagaaatct 780
aacagccatc cagaagtaca tggagatttt ctcaaccagg aacagttaga tctagactcc 840
atgggttatc ctatgttacc atcctccatg ttaggagctg gcatgatctt ggttaattct 900
tttgaagacc cttttggcga catatatggt gaaggtgtct ggagaaaatt cagcagaaga 960
gcttctatcc taaatcgcaa tgttgtcgca ctggcttcat tcaatggaga aaaaaggttt 1020
tttgcatgca caggtttttt tattgaatgg agtggatcta agatgatttt gacatcagcg 1080
agcttggtta gagattctgg tgatgagaac aagattgatg aaaacttgag gattaaagtg 1140
tttcttaaca accaatgcaa agaagggaag ttagagcatt gcaatctaca ttacaacatt 1200
gctctagtca gtgtcaagta ccgtgctctt cgtccattaa atacgagctt tgattgcaag 1260
tcttctagag tagtggctgt agggcgttgc ttcaattctg gcacgttaat ggctactagt 1320
ggtcgtctag ttccttggac aggcacactt gactgccagt tcctcgcacg ttccacgtgt 1380
aaaattacta aggctgggat tggaggccct cttgttaatc ttgatggcaa tgttattggc 1440
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aagccacgct ggtgccgtcc tgaagatgct gaatctgatg atgatgataa gttagctttt 1680
gatgatattg gccagcttca atacagttat attttgggac gtaaagtcaa gctccttcgt 1740
ctcacaatcc ccatatcggt tccgattgtt gaagctaaat ccactgatga gcctggagtt 1800
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actggtctca gtgatgggct taaatcatat ttgtctaaaa gtgtcgcttc tattattttg 180
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Claims (5)
1.亚洲栽培稻与非洲栽培稻种间杂种不育S1座位相关的OgTPR1基因在克服所述种间杂种不育中的应用,所述应用为定点敲除非洲栽培稻中的OgTPR1基因,所述OgTPR1基因的编码序列如SEQ ID NO:1所示。
2.一种创建亚洲栽培稻与非洲栽培稻种间杂种S1座位亲和系的方法,其特征在于,是通过定点敲除OgTPR1,构建亚洲栽培稻与非洲栽培稻种间杂种S1座位亲和系,所述方法包括以下步骤:
(1)针对OgTPR1基因的序列,以一种基因定点编辑***,选择OgTPR1中的1个或多个靶点,构建针对OgTPR1的基因编辑转化载体;
(2)将针对OgTPR1的基因编辑转化载体导入含有OgTPR1的非洲稻受体材料,获得敲除OgTPR1基因功能的植株;
(3)通过PCR扩增包含靶点的目的片段并进行测序分析突变情况,获得相应的OgTPR1基因功能敲除突变体ogtpr1,即获得用于与亚洲稻杂交的杂种S1座位亲和系;
所述OgTPR1基因的编码序列如SEQ ID NO:1所示。
3.根据权利要求2所述的方法,其特征在于,步骤(1)所述的靶点序列如SEQ ID NO:4所示。
4.权利要求2所述创建亚洲栽培稻与非洲栽培稻种间杂种S1座位亲和系的方法在克服亚洲栽培稻与非洲栽培稻的种间杂种不育中的应用。
5.根据权利要求4所述的应用,其特征在于,将权利要求2所述方法创建得到的S1座位亲和系与亚洲栽培稻杂交,获得杂种一代及其后代,这些杂种的花粉和雌配子育性得以恢复正常。
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