CN105063061A - 一种水稻千粒重基因tgw6突变体及其制备方法与应用 - Google Patents
一种水稻千粒重基因tgw6突变体及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于植物生物技术领域,具体涉及一种水稻千粒重基因tgw6突变体及其制备方法与应用。本发明通过设计特定的TGW6位点,利用CRISPR/Cas9技术定点编辑了调控水稻千粒重的TGW6基因,获得了一套具有重要应用价值的水稻tgw6缺失突变体新种质,分别为Cas-tgw6-a、Cas-tgw6b或Cas-tgw6c,该类突变体显著影响水稻的千粒重,提高水稻千粒重5%以上。可以用于水稻的高产、稳产育种。
Description
技术领域
本发明属于植物生物技术领域,具体涉及一种水稻千粒重基因tgw6突变体及其制备方法与应用。
背景技术
水稻(OryzasativaL.)是世界上重要的粮食作物之一,养活着全球半数以上的人口,也是我国近一半人口的主要食物来源。随着人口的不断增长,人们对粮食的需求也越来越大。杂种优势的有效利用对水稻产量的提高起了重要的推动作用,然而,单位面积的水稻产量一直没有太大提升,而且随着耕地面积的减少,水稻的种植面积一直在下降,迫切需要借助新的遗传改良策略来大力提高水稻的产量。近几年,随着几个控制谷粒大小的基因(GS3、SW5和GW8)、粒宽粒重(GW2、GW5)、粒长粒宽的基因(qGLl、qGWl、GS7和qSS7)及千粒重基因(TGW6)等与产量相关基因相继被克隆,部分产量相关基因已经被广泛用于水稻高产品种的培育中。这些产量性状相关基因中,以调控粒长粒重的千粒重基因(Thousand-grainWeight6,TGW6)的遗传力最大,Ishimaru等(2013)的研究发现,Kasalath的tgw6基因能使日本晴在抽穗前的籽粒碳水化合物的积累增加,从而使日本晴产量增加15%却不影响稻米品质;进一步研究发现,Kasalath的tgw6基因在313bp处发生单碱基缺失造成移码突变而使得翻译提前终止不能形成成熟蛋白,从而通过对源器官的多效影响增加千粒重从而使水稻增产。TGW6基因编码吲哚乙酸-葡糖糖水解酶,其功能缺失突变会引起胚乳中吲哚乙酸含量下降,进而使细胞数量增加,粒长变长、粒重增加。
一直以来,科学家都未发现准确、便捷、高效率的植物基因组编辑的方法。最近发现的CRISPR/Cas9系统因其简易和有效性已被广泛应用于包括植物在内的多种生物的基因组编辑中,该系统仅需要短引导RNA和核酸酶就可以对特定生物的靶基因进行定点突变,为生物定点诱变技术发展注入了新的活力。目前CRISPR/Cas9系统已成功在拟南芥、烟草、甜橙、水稻、小麦、高粱、玉米以及苔藓植物地钱等植物中实现了定点基因组编辑,但对水稻育种中有重要价值的产量、品质、育性等关键基因的定向编辑的研究鲜有报道,更缺乏对相关突变体的育种价值评价。
发明内容
为了克服现有技术的缺点和不足,本发明的首要目的在于提供一种水稻千粒重基因tgw6突变体,该突变体为缺失突变体。
本发明的另一目的在于提供上述水稻千粒重基因tgw6突变体的制备方法。
本发明的再一目的在于提供上述水稻千粒重基因tgw6突变体的应用。
一种水稻千粒重基因tgw6突变体,分别为Cas-tgw6a、Cas-tgw6b或Cas-tgw6c;
所述的Cas-tgw6a中,千粒重基因tgw6的氨基酸序列为:
MRNYKTGNLYIADAYMGLMRVGPKGGEATVLAMKADGVPLRFTNGVDIDQVTGDVYFTDSSMNYQRSQHEQVTATKDSTGRLMKYDPRTNQVTVLQSNITYPNGVAMSADRTHLIVALTGPCKLMRHWIRGPKTGKSEPFVDLPGYPDNVRPDGKGGYWIALHREKYELPFGPDSHLVAMRVSAGGKLVQQMRGPKSLRPTEVMERKDGKIYMGNVELPYVGVVKSS;
所述的Cas-tgw6b中,千粒重基因tgw6的氨基酸序列为:
MGRITGRPGERRVRRQRPRPVQRRLRRPHHEVERRGRWLEHLHVQPQLHEKQVRGIDSPHGPDREQMRPPVRPTVSLQNRQPVHRRRLHGIDASWSKRRGGNRASHEG;
所述的Cas-tgw6c中,千粒重基因tgw6的氨基酸序列为:
MRMFKTIDARRSQHLDLGGSLVGPESVAFDGKGRGPYSGVSDGRIMRWNGEAAGWSTYTYSPSYTKNKCAASTLPTVQTESKCGRPLGLRFHYKTGNLYIADAYMGLMRVGPKGGEATVLAMKADGVPLRFTNGVDIDQVTGDVYFTDSSMNYQRSQHEQVTATKDSTGRLMKYDPRTNQVTVLQSNITYPNGVAMSADRTHLIVALTGPCKLMRHWIRGPKTGKSEPFVDLPGYPDNVRPDGKGGYWIALHREKYELPFGPDSHLVAMRVSAGGKLVQQMRGPKSLRPTEVMERKDGKIYMGNVELPYVGVVKSS;
所述的Cas-tgw6a中,千粒重基因tgw6的核苷酸序列为:
ATGAGAAACTACAAAACCGGCAACCTGTACATCGCCGACGCCTACATGGGATTGATGCGAGTTGGTCCAAAAGGCGGGGAGGCAACCGTGCTAGCCATGAAGGCTGATGGCGTGCCACTTCGCTTCACCAATGGGGTGGACATTGATCAGGTTACCGGAGATGTTTATTTCACCGACAGCAGCATGAACTACCAACGATCTCAGCACGAGCAAGTCACGGCGACCAAGGATTCGACCGGACGGCTCATGAAGTATGACCCACGAACTAACCAAGTCACCGTTCTTCAATCCAACATAACCTACCCGAACGGTGTCGCCATGAGCGCTGACCGAACACATCTGATCGTTGCATTGACCGGGCCATGTAAGTTGATGAGGCATTGGATCCGAGGCCCGAAGACTGGCAAATCTGAACCATTTGTTGACCTGCCAGGCTATCCTGATAATGTGAGGCCTGATGGAAAAGGTGGTTATTGGATAGCGCTTCATCGCGAGAAGTATGAGCTTCCCTTTGGTCCGGATAGTCACTTGGTTGCTATGAGGGTTAGTGCTGGTGGGAAGCTGGTTCAACAGATGAGAGGACCAAAGAGCTTGAGGCCAACCGAAGTGATGGAGAGGAAGGATGGCAAAATATACATGGGAAATGTTGAATTGCCGTATGTCGGAGTCGTCAAAAGCAGCTAG;
所述的Cas-tgw6b中,千粒重基因tgw6的核苷酸序列为:
ATGGGGCGGATCACTGGTCGGCCCGGAGAGCGTCGCGTTCGACGGCAAAGGCCGCGGCCCGTACAGCGGCGTCTCCGACGGCCGCATCATGAGGTGGAACGGCGAGGCCGCTGGCTGGAGCACCTACACGTACAGCCCCAGCTACACGAAAAACAAGTGCGCGGCATCGACTCTCCCCACGGTCCAGACCGAGAGCAAATGCGGCCGCCCGTTAGGCCTACGGTTTCACTACAAAACCGGCAACCTGTACATCGCCGACGCCTACATGGGATTGATGCGAGTTGGTCCAAAAGGCGGGGAGGCAACCGTGCTAGCCATGAAGGCTGATGGCGTGCCACTTCGCTTCACCAATGGGGTGGACATTGATCAGGTTACCGGAGATGTTTATTTCACCGACAGCAGCATGAACTACCAACGATCTCAGCACGAGCAAGTCACGGCGACCAAGGATTCGACCGGACGGCTCATGAAGTATGACCCACGAACTAACCAAGTCACCGTTCTTCAATCCAACATAACCTACCCGAACGGTGTCGCCATGAGCGCTGACCGAACACATCTGATCGTTGCATTGACCGGGCCATGTAAGTTGATGAGGCATTGGATCCGAGGCCCGAAGACTGGCAAATCTGAACCATTTGTTGACCTGCCAGGCTATCCTGATAATGTGAGGCCTGATGGAAAAGGTGGTTATTGGATAGCGCTTCATCGCGAGAAGTATGAGCTTCCCTTTGGTCCGGATAGTCACTTGGTTGCTATGAGGGTTAGTGCTGGTGGGAAGCTGGTTCAACAGATGAGAGGACCAAAGAGCTTGAGGCCAACCGAAGTGATGGAGAGGAAGGATGGCAAAATATACATGGGAAATGTTGAATTGCCGTATGTCGGAGTCGTCAAAAGCAGCTAG;
所述的Cas-tgw6c中,千粒重基因tgw6的核苷酸序列为:
ATGAGAATGTTCAAGACCATTGACGCCCGGCGGAGCCAGCATCTGGACCTCGGCGGATCACTGGTCGGCCCGGAGAGCGTCGCGTTCGACGGCAAAGGCCGCGGCCCGTACAGCGGCGTCTCCGACGGCCGCATCATGAGGTGGAACGGCGAGGCCGCTGGCTGGAGCACCTACACGTACAGCCCCAGCTACACGAAAAACAAGTGCGCGGCATCGACTCTCCCCACGGTCCAGACCGAGAGCAAATGCGGCCGCCCGTTAGGCCTACGGTTTCACTACAAAACCGGCAACCTGTACATCGCCGACGCCTACATGGGATTGATGCGAGTTGGTCCAAAAGGCGGGGAGGCAACCGTGCTAGCCATGAAGGCTGATGGCGTGCCACTTCGCTTCACCAATGGGGTGGACATTGATCAGGTTACCGGAGATGTTTATTTCACCGACAGCAGCATGAACTACCAACGATCTCAGCACGAGCAAGTCACGGCGACCAAGGATTCGACCGGACGGCTCATGAAGTATGACCCACGAACTAACCAAGTCACCGTTCTTCAATCCAACATAACCTACCCGAACGGTGTCGCCATGAGCGCTGACCGAACACATCTGATCGTTGCATTGACCGGGCCATGTAAGTTGATGAGGCATTGGATCCGAGGCCCGAAGACTGGCAAATCTGAACCATTTGTTGACCTGCCAGGCTATCCTGATAATGTGAGGCCTGATGGAAAAGGTGGTTATTGGATAGCGCTTCATCGCGAGAAGTATGAGCTTCCCTTTGGTCCGGATAGTCACTTGGTTGCTATGAGGGTTAGTGCTGGTGGGAAGCTGGTTCAACAGATGAGAGGACCAAAGAGCTTGAGGCCAACCGAAGTGATGGAGAGGAAGGATGGCAAAATATACATGGGAAATGTTGAATTGCCGTATGTCGGAGTCGTCAAAAGCAGCTAG;
所述的水稻千粒重基因tgw6突变体的制备方法,包含如下步骤:
(1)引导RNA(guideRNA,gRNA)靶点序列设计与选择
根据调控水稻粒长及千粒重基因TGW6的基因组序列,设计、选择并合成3个TGW6引导RNA靶点序列;
(2)三靶点CRISPR/Cas9-gRNA载体的构建
将步骤(1)中合成的引导RNA靶点序列的寡核苷酸链退火形成双链,然后分别与BsaI酶切后的pYL-U3-gRNA、pYL-U6a-gRNA和pYL-U6b-gRNA连接,得到三个gRNA表达盒;然后通过Goldengatecloning的方法将gRNA表达盒依次装载到CRISPR/Cas9载体上,得到三靶点CRISPR/Cas9-gRNA载体;
(3)农杆菌介导水稻愈伤遗传转化
将步骤(2)构建好的三靶点CRISPR/Cas9-gRNA载体转化到水稻中,分别获得不含CRISPR元件T-DNA成分的纯合tgw6突变体Cas-tgw6a、Cas-tgw6b或Cas-tgw6c;
步骤(1)中所述的3个TGW6引导RNA靶点序列的寡核苷酸序列分别为:
TGW6U3-T1-F:5’-GGCAGCCAGCATCTGGACCTCGG-3’;
TGW6U3-T1-R:5’-AAACCCGAGGTCCAGATGCTGGC-3’;
TGW6U6a-T2-F:5’-GCCGGCTACAGCCATGAGAAGCA-3’;
TGW6U6a-T2-R:5’-AAACTGCTTCTCATGGCTGTAGC-3’;
TGW6U6b-T3-F:5’-GTTGAGGCAAGCGGCGACCGCGG-3’;
TGW6U6b-T3-R:5’-AAACCCGCGGTCGCCGCTTGCCT-3’;
步骤(2)中所述的Goldengatecloning的引物优选为:
U3-T1-F:5’-TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG-3’;
U3-T1-R:5’-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3’;
U6a-T2-F:5’-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3’;
U6a-T2-R:5’-AGCGTGGGTCTCGTCTTGGTCCATCCACTCCAAGCTC-3’;
U6b-T3-F:5’-TTCAGAGGTCTCTAAGACACTGGAATCGGCAGCAAAGG-3’;
U6b-T3-R:5’-AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC-3’;
步骤(2)中所述的CRISPR/Cas9载体优选为pYLCRISPR/Cas9Pubi-H;
步骤(3)中所述的水稻的品系为H447;
所述的H447为R819/玉香占//R819BC2F7代稳定品系,即为先将R819与玉香占杂交,再将杂交后代与R819回交两次后再自交7代后所得的稳定品系;
所述的水稻千粒重基因tgw6突变体在水稻种植领域中的应用。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明通过设计特定的tgw6位点,利用CRISPR/Cas9技术定点编辑了调控水稻千粒重的TGW6基因,获得了一套具有重要应用价值的水稻tgw6缺失突变体新种质,这些突变体可以用于水稻的高产、稳产育种。
(2)本发明基于CRIPSR/Cas9技术的千粒重基因tgw6突变体创建的成功实施,为快速创建稻米品质(fgr、Chalk5)等生产上有重要应用价值的水稻优异新种质提供了重要参考,并有望为水稻种质资源创新提供了安全、高效的新途径,具有重要的理论和实践意义。
附图说明
图1是TGW6基因靶点序列引物设计及靶点组装示意图,其中,A:3个靶点在TGW6基因上的位置;B:各gRNA表达盒在载体pYLCRISPR/Cas9Pubi-H中的组装方式,黑色方框代表靶序列引物所在位置。
图2是利用TGW6基因靶位点附近的特异引物对突变体的检测结果,其中,M:1kbDNAladder;1:水稻材料H447(野生型);2~22:tgw6突变体。
图3是部分tgw6纯合缺失突变体的突变点的测序分析图,其中“….”示为省略序列,“----”示为缺失序列;WT为水稻材料H447,4、18及8分别为tgw6缺失突变体Cas-tgw6a、Cas-tgw6b及Cas-tgw6c。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
载体pYL-U3-gRNA、pYL-U6a-gRNA和pYL-U6b-gRNA以及载体pYLCRISPR/Cas9Pubi-H均由华南农业大学生命科学学院提供(MaX,ZhangQ,ZhuQ,LiuW,ChenY,QiuR,WangB,YangZ.2015.ArobustCRISPR/Cas9systemforconvenient,high-efficiencymultiplexgenomeeditinginmonocotanddicotplants.MolPlant.doi:10.1016/j.molp.2015.04.007);
水稻品系H447为R819/玉香占//R819BC2F7代稳定品系,即为先将R819与玉香占杂交,再将杂交后代与R819回交两次后再自交7代后所得的稳定品系;
实施例1基于CRISPR/Cas9技术的水稻千粒重基因tgw6缺失突变体的创建
(1)引导RNA(guideRNA,gRNA)靶点序列的设计根据调控水稻粒长及千粒重基因TGW6的基因组序列(GenBank:AB513135.1),设计3个靶向千粒重基因TGW6的gRNA。20nt的寡核苷酸gRNA靶点序列按A/G(N)20NGG序列进行设计,同时将设计好的gRNA靶点序列进行水稻基因组数据库比对以排除非特异性的靶切位点,具体寡核苷酸序列见表1和图1。
表1gRNA靶点的寡核苷酸序列
(2)三靶点CRISPR/Cas9-gRNA载体的构建
参照Ma等人(MaX,ZhangQ,ZhuQ,LiuW,ChenY,QiuR,WangB,YangZ.2015.ArobustCRISPR/Cas9systemforconvenient,high-efficiencymultiplexgenomeeditinginmonocotanddicotplants.MolPlant.doi:10.1016/j.molp.2015.04.007)的方法,取等量的上游与下游gRNA寡核苷酸链(步骤(1))混合(终浓度1μM),90℃30sec,移至室温冷却完成退火形成双链接头;取pYL-U3-gRNA、pYL-U6a-gRNA及pYL-U6b-gRNA质粒各1μg,在20μL反应体系中用10UBsaI(NEB公司)酶切20min,然后将酶切过的pYL-U3/U6a/U6b-gRNA载体与各自所对应的双链接头利用T4DNAligase22℃连接30min后,得到3个gRNA表达盒(U3、U6a、U6b),其中,具体连接体系为:
然后分别用表2中所列各对引物对各个gRNA表达盒(U3、U6a、U6b)进行扩增:
表2扩增各靶点gRNA表达盒的引物
具体的扩增体系为:
然后进行以下反应:95℃1min;95℃15sec、55℃15sec、68℃20sec10循环;95℃15sec、60℃15sec68℃20sec17~20循环。
将这三个靶点gDNAPCR产物混合后进行PCR产物的纯化,纯化产物经20UBsaI37℃酶切30min并纯化后,将其与经BsaI酶切的pYLCRISPR/Cas9Pubi-H片段利用T4DNA连接酶20℃连接约2h,具体连接体系为:
最后转化DH5α感受态细胞并挑取阳性单克隆(三个片段的连接顺序是U3-U6a-U6b)后进行测序验证(由Invitrogen公司完成),得到三靶点CRISPR/Cas9-gRNA载体。
实施例2
(1)农杆菌介导水稻愈伤遗传转化
将实施例1组装好的CRISPR/Cas9-gRNA载体通过电击转化到农杆菌EHA105中。PCR检测(引物为:hptF:5’-TCCGGAGCCTCCGCTCGAAGTAG-3’、hptR:5’-CTGAACTCACCGCGACGTCTGTC-3’)为阳性的克隆用于侵染水稻材料H447(R819/玉针香//R819的BC2F7)的愈伤组织,侵染方法参照Hiei等(1994)的方法进行,获得转基因水稻植株。
(2)水稻基因组DNA提取及突变体的PCR检测及测序分析
采用CTAB法提取步骤(1)中获得的水稻植株的基因组DNA,对潮霉素基因检测为阳性的植株利用引物Cas9-TGW6testF和Cas9-TGW6testR对tgw6突变位点进行PCR检测,其中鉴定引物为:
Cas9-TGW6-test-F:5’-CAACCAAACCAAAGCCTGC-3’;
Cas9-TGW6-test-R:5’-CCAATGCCTCATCAACTTAC-3’;
PCR扩增体系为:
反应条件为:94℃预变性3min;94℃变性30sec,55℃退火30sec,68℃延伸60sec,32个循环;68℃延伸5min;
扩增产物经1%琼脂糖凝胶电泳(电泳缓冲液1×TAE),BIORAD凝胶成像系统观察、照相。结果表明,扩增产物中多为片段缺失纯合体,也有部分双等位杂合突变体存在(图2)。对PCR产物进行的测序结果分析表明,tgw6突变频率在90%以上,其中50%多为片段缺失纯合体,有40%左右是双等位杂合突变体(图3)。
(4)无转基因成分tgw6突变体的获得及相关性状调查
将T0代转基因植株播种成苗,苗期检测潮霉素基因的有无,将不含潮霉素基因而且tgw6基因位点有缺失的纯合个体(编号为18、8的缺失纯合体,分别命名为Cas-tgw6b、Cas-tgw6c)种植至收获T1代种子,并对T1代种子进行千粒重调查:Cas-tgw6b、Cas-tgw6c显著影响了水稻的千粒重,提高了千粒重5%以上。
对于tgw6基因位点双等位杂合个体(编号为4),需自交后去除潮霉素基因影响,得到纯合缺失突变体,命名为Cas-tgw6a,然后计算其千粒重,同样的,Cas-tgw6a显著影响了水稻的千粒重,提高了千粒重5%以上。
利用引物Cas9-TGW6testF和Cas9-TGW6testR对突变体Cas-tgw6a、Cas-tgw6b和Cas-tgw6c的tgw6突变位点进行PCR检测并测序(图3);
其中,突变体Cas-tgw6a中,千粒重基因tgw6的核苷酸序列为:
ATGAGAAACTACAAAACCGGCAACCTGTACATCGCCGACGCCTACATGGGATTGATGCGAGTTGGTCCAAAAGGCGGGGAGGCAACCGTGCTAGCCATGAAGGCTGATGGCGTGCCACTTCGCTTCACCAATGGGGTGGACATTGATCAGGTTACCGGAGATGTTTATTTCACCGACAGCAGCATGAACTACCAACGATCTCAGCACGAGCAAGTCACGGCGACCAAGGATTCGACCGGACGGCTCATGAAGTATGACCCACGAACTAACCAAGTCACCGTTCTTCAATCCAACATAACCTACCCGAACGGTGTCGCCATGAGCGCTGACCGAACACATCTGATCGTTGCATTGACCGGGCCATGTAAGTTGATGAGGCATTGGATCCGAGGCCCGAAGACTGGCAAATCTGAACCATTTGTTGACCTGCCAGGCTATCCTGATAATGTGAGGCCTGATGGAAAAGGTGGTTATTGGATAGCGCTTCATCGCGAGAAGTATGAGCTTCCCTTTGGTCCGGATAGTCACTTGGTTGCTATGAGGGTTAGTGCTGGTGGGAAGCTGGTTCAACAGATGAGAGGACCAAAGAGCTTGAGGCCAACCGAAGTGATGGAGAGGAAGGATGGCAAAATATACATGGGAAATGTTGAATTGCCGTATGTCGGAGTCGTCAAAAGCAGCTAG;
突变体Cas-tgw6a,千粒重基因tgw6的氨基酸序列为:
MRNYKTGNLYIADAYMGLMRVGPKGGEATVLAMKADGVPLRFTNGVDIDQVTGDVYFTDSSMNYQRSQHEQVTATKDSTGRLMKYDPRTNQVTVLQSNITYPNGVAMSADRTHLIVALTGPCKLMRHWIRGPKTGKSEPFVDLPGYPDNVRPDGKGGYWIALHREKYELPFGPDSHLVAMRVSAGGKLVQQMRGPKSLRPTEVMERKDGKIYMGNVELPYVGVVKSS;
突变体Cas-tgw6b中,千粒重基因tgw6的核苷酸序列为:
ATGGGGCGGATCACTGGTCGGCCCGGAGAGCGTCGCGTTCGACGGCAAAGGCCGCGGCCCGTACAGCGGCGTCTCCGACGGCCGCATCATGAGGTGGAACGGCGAGGCCGCTGGCTGGAGCACCTACACGTACAGCCCCAGCTACACGAAAAACAAGTGCGCGGCATCGACTCTCCCCACGGTCCAGACCGAGAGCAAATGCGGCCGCCCGTTAGGCCTACGGTTTCACTACAAAACCGGCAACCTGTACATCGCCGACGCCTACATGGGATTGATGCGAGTTGGTCCAAAAGGCGGGGAGGCAACCGTGCTAGCCATGAAGGCTGATGGCGTGCCACTTCGCTTCACCAATGGGGTGGACATTGATCAGGTTACCGGAGATGTTTATTTCACCGACAGCAGCATGAACTACCAACGATCTCAGCACGAGCAAGTCACGGCGACCAAGGATTCGACCGGACGGCTCATGAAGTATGACCCACGAACTAACCAAGTCACCGTTCTTCAATCCAACATAACCTACCCGAACGGTGTCGCCATGAGCGCTGACCGAACACATCTGATCGTTGCATTGACCGGGCCATGTAAGTTGATGAGGCATTGGATCCGAGGCCCGAAGACTGGCAAATCTGAACCATTTGTTGACCTGCCAGGCTATCCTGATAATGTGAGGCCTGATGGAAAAGGTGGTTATTGGATAGCGCTTCATCGCGAGAAGTATGAGCTTCCCTTTGGTCCGGATAGTCACTTGGTTGCTATGAGGGTTAGTGCTGGTGGGAAGCTGGTTCAACAGATGAGAGGACCAAAGAGCTTGAGGCCAACCGAAGTGATGGAGAGGAAGGATGGCAAAATATACATGGGAAATGTTGAATTGCCGTATGTCGGAGTCGTCAAAAGCAGCTAG;
突变体Cas-tgw6b中,千粒重基因tgw6的氨基酸序列为:MGRITGRPGERRVRRQRPRPVQRRLRRPHHEVERRGRWLEHLHVQPQLHEKQVRGIDSPHGPDREQMRPPVRPTVSLQNRQPVHRRRLHGIDASWSKRRGGNRASHEG;
突变体Cas-tgw6c中,千粒重基因tgw6的核苷酸序列为:
ATGAGAATGTTCAAGACCATTGACGCCCGGCGGAGCCAGCATCTGGACCTCGGCGGATCACTGGTCGGCCCGGAGAGCGTCGCGTTCGACGGCAAAGGCCGCGGCCCGTACAGCGGCGTCTCCGACGGCCGCATCATGAGGTGGAACGGCGAGGCCGCTGGCTGGAGCACCTACACGTACAGCCCCAGCTACACGAAAAACAAGTGCGCGGCATCGACTCTCCCCACGGTCCAGACCGAGAGCAAATGCGGCCGCCCGTTAGGCCTACGGTTTCACTACAAAACCGGCAACCTGTACATCGCCGACGCCTACATGGGATTGATGCGAGTTGGTCCAAAAGGCGGGGAGGCAACCGTGCTAGCCATGAAGGCTGATGGCGTGCCACTTCGCTTCACCAATGGGGTGGACATTGATCAGGTTACCGGAGATGTTTATTTCACCGACAGCAGCATGAACTACCAACGATCTCAGCACGAGCAAGTCACGGCGACCAAGGATTCGACCGGACGGCTCATGAAGTATGACCCACGAACTAACCAAGTCACCGTTCTTCAATCCAACATAACCTACCCGAACGGTGTCGCCATGAGCGCTGACCGAACACATCTGATCGTTGCATTGACCGGGCCATGTAAGTTGATGAGGCATTGGATCCGAGGCCCGAAGACTGGCAAATCTGAACCATTTGTTGACCTGCCAGGCTATCCTGATAATGTGAGGCCTGATGGAAAAGGTGGTTATTGGATAGCGCTTCATCGCGAGAAGTATGAGCTTCCCTTTGGTCCGGATAGTCACTTGGTTGCTATGAGGGTTAGTGCTGGTGGGAAGCTGGTTCAACAGATGAGAGGACCAAAGAGCTTGAGGCCAACCGAAGTGATGGAGAGGAAGGATGGCAAAATATACATGGGAAATGTTGAATTGCCGTATGTCGGAGTCGTCAAAAGCAGCTAG;
突变体Cas-tgw6c中,千粒重基因tgw6的氨基酸序列为:
MRMFKTIDARRSQHLDLGGSLVGPESVAFDGKGRGPYSGVSDGRIMRWNGEAAGWSTYTYSPSYTKNKCAASTLPTVQTESKCGRPLGLRFHYKTGNLYIADAYMGLMRVGPKGGEATVLAMKADGVPLRFTNGVDIDQVTGDVYFTDSSMNYQRSQHEQVTATKDSTGRLMKYDPRTNQVTVLQSNITYPNGVAMSADRTHLIVALTGPCKLMRHWIRGPKTGKSEPFVDLPGYPDNVRPDGKGGYWIALHREKYELPFGPDSHLVAMRVSAGGKLVQQMRGPKSLRPTEVMERKDGKIYMGNVELPYVGVVKSS。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种水稻千粒重基因tgw6突变体,其特征在于:
所述的水稻千粒重基因tgw6突变体分别为Cas-tgw6a、Cas-tgw6b或Cas-tgw6c;
所述的Cas-tgw6a中,千粒重基因tgw6的氨基酸序列为:
MRNYKTGNLYIADAYMGLMRVGPKGGEATVLAMKADGVPLRFTNGVDIDQVTGDVYFTDSSMNYQRSQHEQVTATKDSTGRLMKYDPRTNQVTVLQSNITYPNGVAMSADRTHLIVALTGPCKLMRHWIRGPKTGKSEPFVDLPGYPDNVRPDGKGGYWIALHREKYELPFGPDSHLVAMRVSAGGKLVQQMRGPKSLRPTEVMERKDGKIYMGNVELPYVGVVKSS;
所述的Cas-tgw6b中,千粒重基因tgw6的氨基酸序列为:MGRITGRPGERRVRRQRPRPVQRRLRRPHHEVERRGRWLEHLHVQPQLHEKQVRGIDSPHGPDREQMRPPVRPTVSLQNRQPVHRRRLHGIDASWSKRRGGNRASHEG;
所述的Cas-tgw6c中,千粒重基因tgw6的氨基酸序列为:
MRMFKTIDARRSQHLDLGGSLVGPESVAFDGKGRGPYSGVSDGRIMRWNGEAAGWSTYTYSPSYTKNKCAASTLPTVQTESKCGRPLGLRFHYKTGNLYIADAYMGLMRVGPKGGEATVLAMKADGVPLRFTNGVDIDQVTGDVYFTDSSMNYQRSQHEQVTATKDSTGRLMKYDPRTNQVTVLQSNITYPNGVAMSADRTHLIVALTGPCKLMRHWIRGPKTGKSEPFVDLPGYPDNVRPDGKGGYWIALHREKYELPFGPDSHLVAMRVSAGGKLVQQMRGPKSLRPTEVMERKDGKIYMGNVELPYVGVVKSS。
2.根据权利要求1所述的水稻千粒重基因tgw6突变体,其特征在于:
所述的Cas-tgw6a中,千粒重基因tgw6的核苷酸序列为:
ATGAGAAACTACAAAACCGGCAACCTGTACATCGCCGACGCCTACATGGGATTGATGCGAGTTGGTCCAAAAGGCGGGGAGGCAACCGTGCTAGCCATGAAGGCTGATGGCGTGCCACTTCGCTTCACCAATGGGGTGGACATTGATCAGGTTACCGGAGATGTTTATTTCACCGACAGCAGCATGAACTACCAACGATCTCAGCACGAGCAAGTCACGGCGACCAAGGATTCGACCGGACGGCTCATGAAGTATGACCCACGAACTAACCAAGTCACCGTTCTTCAATCCAACATAACCTACCCGAACGGTGTCGCCATGAGCGCTGACCGAACACATCTGATCGTTGCATTGACCGGGCCATGTAAGTTGATGAGGCATTGGATCCGAGGCCCGAAGACTGGCAAATCTGAACCATTTGTTGACCTGCCAGGCTATCCTGATAATGTGAGGCCTGATGGAAAAGGTGGTTATTGGATAGCGCTTCATCGCGAGAAGTATGAGCTTCCCTTTGGTCCGGATAGTCACTTGGTTGCTATGAGGGTTAGTGCTGGTGGGAAGCTGGTTCAACAGATGAGAGGACCAAAGAGCTTGAGGCCAACCGAAGTGATGGAGAGGAAGGATGGCAAAATATACATGGGAAATGTTGAATTGCCGTATGTCGGAGTCGTCAAAAGCAGCTAG。
3.根据权利要求1所述的水稻千粒重基因tgw6突变体,其特征在于:
所述的Cas-tgw6b中,千粒重基因tgw6的核苷酸序列为:
ATGGGGCGGATCACTGGTCGGCCCGGAGAGCGTCGCGTTCGACGGCAAAGGCCGCGGCCCGTACAGCGGCGTCTCCGACGGCCGCATCATGAGGTGGAACGGCGAGGCCGCTGGCTGGAGCACCTACACGTACAGCCCCAGCTACACGAAAAACAAGTGCGCGGCATCGACTCTCCCCACGGTCCAGACCGAGAGCAAATGCGGCCGCCCGTTAGGCCTACGGTTTCACTACAAAACCGGCAACCTGTACATCGCCGACGCCTACATGGGATTGATGCGAGTTGGTCCAAAAGGCGGGGAGGCAACCGTGCTAGCCATGAAGGCTGATGGCGTGCCACTTCGCTTCACCAATGGGGTGGACATTGATCAGGTTACCGGAGATGTTTATTTCACCGACAGCAGCATGAACTACCAACGATCTCAGCACGAGCAAGTCACGGCGACCAAGGATTCGACCGGACGGCTCATGAAGTATGACCCACGAACTAACCAAGTCACCGTTCTTCAATCCAACATAACCTACCCGAACGGTGTCGCCATGAGCGCTGACCGAACACATCTGATCGTTGCATTGACCGGGCCATGTAAGTTGATGAGGCATTGGATCCGAGGCCCGAAGACTGGCAAATCTGAACCATTTGTTGACCTGCCAGGCTATCCTGATAATGTGAGGCCTGATGGAAAAGGTGGTTATTGGATAGCGCTTCATCGCGAGAAGTATGAGCTTCCCTTTGGTCCGGATAGTCACTTGGTTGCTATGAGGGTTAGTGCTGGTGGGAAGCTGGTTCAACAGATGAGAGGACCAAAGAGCTTGAGGCCAACCGAAGTGATGGAGAGGAAGGATGGCAAAATATACATGGGAAATGTTGAATTGCCGTATGTCGGAGTCGTCAAAAGCAGCTAG。
4.根据权利要求1所述的水稻千粒重基因tgw6突变体,其特征在于:
所述的Cas-tgw6c中,千粒重基因tgw6的核苷酸序列为:
ATGAGAATGTTCAAGACCATTGACGCCCGGCGGAGCCAGCATCTGGACCTCGGCGGATCACTGGTCGGCCCGGAGAGCGTCGCGTTCGACGGCAAAGGCCGCGGCCCGTACAGCGGCGTCTCCGACGGCCGCATCATGAGGTGGAACGGCGAGGCCGCTGGCTGGAGCACCTACACGTACAGCCCCAGCTACACGAAAAACAAGTGCGCGGCATCGACTCTCCCCACGGTCCAGACCGAGAGCAAATGCGGCCGCCCGTTAGGCCTACGGTTTCACTACAAAACCGGCAACCTGTACATCGCCGACGCCTACATGGGATTGATGCGAGTTGGTCCAAAAGGCGGGGAGGCAACCGTGCTAGCCATGAAGGCTGATGGCGTGCCACTTCGCTTCACCAATGGGGTGGACATTGATCAGGTTACCGGAGATGTTTATTTCACCGACAGCAGCATGAACTACCAACGATCTCAGCACGAGCAAGTCACGGCGACCAAGGATTCGACCGGACGGCTCATGAAGTATGACCCACGAACTAACCAAGTCACCGTTCTTCAATCCAACATAACCTACCCGAACGGTGTCGCCATGAGCGCTGACCGAACACATCTGATCGTTGCATTGACCGGGCCATGTAAGTTGATGAGGCATTGGATCCGAGGCCCGAAGACTGGCAAATCTGAACCATTTGTTGACCTGCCAGGCTATCCTGATAATGTGAGGCCTGATGGAAAAGGTGGTTATTGGATAGCGCTTCATCGCGAGAAGTATGAGCTTCCCTTTGGTCCGGATAGTCACTTGGTTGCTATGAGGGTTAGTGCTGGTGGGAAGCTGGTTCAACAGATGAGAGGACCAAAGAGCTTGAGGCCAACCGAAGTGATGGAGAGGAAGGATGGCAAAATATACATGGGAAATGTTGAATTGCCGTATGTCGGAGTCGTCAAAAGCAGCTAG。
5.权利要求1~4任一项所述的水稻千粒重基因tgw6突变体的制备方法,其特征在于包含如下步骤:
(1)引导RNA靶点序列设计与选择
根据调控水稻粒长及千粒重基因TGW6的基因组序列,设计、选择并合成3个TGW6引导RNA靶点序列;
(2)三靶点CRISPR/Cas9-gRNA载体的构建
将步骤(1)中合成的引导RNA靶点序列的寡核苷酸链退火形成双链,然后分别与BsaI酶切后的pYL-U3-gRNA、pYL-U6a-gRNA和pYL-U6b-gRNA连接,得到三个gRNA表达盒;然后通过Goldengatecloning的方法将gRNA表达盒依次装载到CRISPR/Cas9载体上,得到三靶点CRISPR/Cas9-gRNA载体;
(3)农杆菌介导水稻愈伤遗传转化
将步骤(2)构建好的三靶点CRISPR/Cas9-gRNA载体转化到水稻中,分别获得不含CRISPR元件T-DNA成分的纯合tgw6突变体Cas-tgw6a、Cas-tgw6b或Cas-tgw6c。
6.根据权利要求5所述的水稻千粒重基因tgw6突变体的制备方法,其特征在于:
步骤(1)中所述的3个TGW6引导RNA靶点序列的寡核苷酸序列分别为:
TGW6U3-T1-F:5’-GGCAGCCAGCATCTGGACCTCGG-3’;
TGW6U3-T1-R:5’-AAACCCGAGGTCCAGATGCTGGC-3’;
TGW6U6a-T2-F:5’-GCCGGCTACAGCCATGAGAAGCA-3’;
TGW6U6a-T2-R:5’-AAACTGCTTCTCATGGCTGTAGC-3’;
TGW6U6b-T3-F:5’-GTTGAGGCAAGCGGCGACCGCGG-3’;
TGW6U6b-T3-R:5’-AAACCCGCGGTCGCCGCTTGCCT-3’。
7.根据权利要求5所述的水稻千粒重基因tgw6突变体的制备方法,其特征在于:
步骤(2)中所述的Goldengatecloning的引物为:
U3-T1-F:5’-TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG-3’;
U3-T1-R:5’-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3’;
U6a-T2-F:5’-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3’;
U6a-T2-R:5’-AGCGTGGGTCTCGTCTTGGTCCATCCACTCCAAGCTC-3’;
U6b-T3-F:5’-TTCAGAGGTCTCTAAGACACTGGAATCGGCAGCAAAGG-3’;
U6b-T3-R:5’-AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC-3’。
8.根据权利要求5所述的水稻千粒重基因tgw6突变体的制备方法,其特征在于:
步骤(2)中所述的CRISPR/Cas9载体为pYLCRISPR/Cas9Pubi-H。
9.根据权利要求5所述的水稻千粒重基因tgw6突变体的制备方法,其特征在于:
步骤(3)中所述的水稻的品系为H447;
所述的H447为R819/玉香占//R819BC2F7代稳定品系。
10.权利要求1~4任一项所述的水稻千粒重基因tgw6突变体在水稻种植领域中的应用。
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