CN104004782A - 一种延长水稻生育期的育种方法 - Google Patents
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Abstract
本发明涉及一种延长水稻生育期的育种方法，包含以下步骤：在水稻生育期决定基因Ehd3外显子区选取靶标片段并构建植物CRISPR/Cas9打靶重组载体，导入水稻细胞并再生成苗；利用载体中的表达框实现对水稻细胞中Ehd3外显子区DNA的剪切，从而引起水稻细胞的自我修复；通过对再生株系基因组目标片段的测序，获取携带两个等位Ehd3基因同时发生功能缺失突变的株系，经过表型鉴定，确认再生植株生育期的延长。实验表明，本方法可快速获得生育期延长的水稻材料。
Description
技术领域
本发明属于水稻生物技术育种领域，具体涉及一种延长水稻生育期的育种方法。
背景技术
水稻是我国的第一大粮食作物，目前水稻种植面积为3000万hm2左右，其中杂交水稻种植面积占总水稻面积的57％，杂交稻制(繁)种面积18万hm2左右。杂交稻种子质量的优劣是实现水稻优质、高产和高效生产的基础，杂交稻种子生产水平高低直接关系到我国粮食安全。
杂交水稻种子生产为水稻的异交栽培，即利用杂交稻雄性不育系作母本，雄性不育恢复系作父本，按照一定的行比相间种植，使双亲能够花期相遇，不育系接受恢复系的花粉而授精结实作为杂交种子应用于生产。同时为了保证下年的杂交种子生产，还要利用杂交稻雄性不育系作母本，雄性不育保持系作父本，按照一定的行比相间种植，使其花期相遇，不育系接受保持系的花粉而授精结实作为雄性不育系种子繁殖应用于下年制种。
目前我国的杂交水稻制种技术与研制初期并没有根本的改进，杂交稻制种程序复杂而严格，仍然采用不育系和恢复系差期播种和移栽、人工赶粉、分别仔细收获等，才能防止机械混杂，劳动强度大，因此，杂交稻制种效率不高。并且杂交稻制种产量受技术人员的经验丰富与否及气候条件好坏影响极大，因此，制种效益也不稳定。由于目前我国农村劳动力结构发生了变化，青壮年外出务工，妇女与老年劳力在家务农。因此，积极发展杂交稻机械化制种技术，实现杂交稻制种规模化，是提高我国杂交稻制种效益的必由之路。
不育系和恢复系和生育期一致是实现机械化混播制种的关键。但目前目前绝大多数强优杂交稻组合父母本生育期不一致，主要表现在不育系的生育期短于恢复系，严重的制约了杂交水稻制种的机械化进程。因此，发展可快速定向延长现有水稻不育系生育期的育种方法，对杂交水稻机械化混播制种的推进将有重要意义。
发明内容
本发明提供了一种延长水稻生育期的育种方法，其特征在于，所述方法包括如下步骤：
步骤1，在水稻生育期决定基因Ehd3(LOC_Os08g01420)外显子区选取靶标片段，所述靶标片段的双链结构中的一条链具有5’-(N)X-NGG-3’结构，(N)X代表数目为X的一段碱基序列{N1,N2……Nx}，X代表碱基的数量，N1,N2……Nx中的每一个表示A、G、C、T中的任意一个，NGG中的N也表示A、T、G、C中的一种；
步骤2，利用所述靶标片段，构建用于水稻Ehd3基因打靶的CRISPR/Cas9重组载体，其中所述重组载体中包含具有所述靶标片段的向导RNA表达框和Cas9核酸酶表达框；
步骤3，将步骤2所获的所述重组载体导入水稻细胞，诱导所述向导RNA表达框和所述Cas9核酸酶表达框在水稻细胞中共同表达；
步骤4，剪切Ehd3基因的双链靶标片段，再通过水稻细胞自身的DNA修复功能，在靶标位点随机插入或缺失碱基，实现细胞内Ehd3基因的功能缺失突变；
步骤5，用导入所述重组载体的水稻细胞再生水稻植株；
步骤6，对再生的水稻植株中Ehd3基因包含靶标片段的DNA区段测序；
步骤7，选择两个等位Ehd3基因都出现功能缺失突变的再生株系，进行表型鉴定，观察再生株系子代的生育期，挑取生育期显著推迟的株系，作为所创制的生育期推迟的水稻材料。
优选地，X为19或20。
优选地，所述向导RNA表达框和所述Cas9核酸酶表达框均能够在水稻细胞中表达。
优选地，所述向导RNA表达框的核苷酸序列如Seq ID No.1所示；所述Cas9核酸酶表达框的核苷酸序列如Seq ID No.2所示。
优选地，所述向导RNA表达框包括：水稻U6启动子，其核苷酸序列如Seq ID No.1第1至246位所示；结构特征为(N)X的靶标序列和人工合成的sgRNA骨架序列，其核苷酸序列如Seq ID No.1第267至350位所示；以及Poly-T终止子，其核苷酸序列如Seq ID No.1第351至358位所示。
优选地，所述Cas9核酸酶表达框包括：玉米ZmUBI启动子，其核苷酸序列如Seq ID No.2第1至2031位所示；植物偏好密码子改造后的Cas9编码序列，其核苷酸序列如Seq ID No.2第2034至6305位所示；以及tNOS终止子，其核苷酸序列如Seq ID No.2第6347至6599位所示。
在所述Ehd3基因外显子上，具有所述(N)X-NGG-3’结构的片段可选为靶标的共有75个。
构建用于水稻Ehd3基因打靶的CRISPR/Cas9重组载体是按照靶标序列的核酸排列顺序进行的，其中所述重组载体中包括CRISPR RNA(crRNA)序列，其为所述靶标区段5’-(N)X-NGG-3’中的(N)X或与之互补的序列。
所述Cas9核酸酶表达框与向导RNA表达框位于同一载体中。
在所述步骤3中，将步骤2所获重组载体导入水稻细胞，从而使细胞同时含有向导RNA表达框和Cas9核酸酶表达框；在向导RNA和Cas9核酸酶的共同作用下，能够对Ehd3基因的双链的所述靶标片段进行剪切，再通过水稻细胞自身的DNA修复功能，最终实现细胞内Ehd3基因靶标片段的随机插入和/或随机缺失。
所述方法中，将重组载体导入水稻细胞所采用的方法为PEG介导的水稻原生质体瞬时转化或农杆菌介导的水稻愈伤组织稳定转化。这两种方法最适合用于本发明的育种方法，尤其是前者。
在步骤4中，再生植物的方法为细胞或组织经过组织培养，获得植株。
在步骤5中，还可以通过基因组PCR方法克隆再生植株中Ehd3基因包含靶标片段的DNA区段，并对扩增产物测序。所述基因组PCR方法为，针对包含靶标片段的基因组区域，设计位点特异性引物，以再生植株的基因组DNA为模板，扩增所述包含靶标片段的基因组区域。扩增产物测序是指，对PCR产物中的目的条带测序。
在步骤6中，两个等位Ehd3基因都出现功能缺失突变是指测序结果在Ehd3基因靶标位点出现两种功能缺失突变序列，且没有出现野生型序列；其中所述功能缺失突变序列指的是正常Ehd3编码序列在靶标位点出现终止子或阅读框移位。
在步骤7中，观察再生株系及自交子代的生育期，挑取生育期推迟2周以上的株系，该材料即为所改良的生育期延长的水稻材料。
在一种实现方式中，所采用的向导RNA表达框具体为(Seq ID No.1)：ggatcatgaaccaacggcctggctgtatttggtggttgtgtagggagatggggagaagaaaagcccgattctcttcgctgtgatgggctggatgcatgcgggggagcgggaggcccaagtacgtgcacggtgagcggcccacagggcgagtgtgagcgcgagaggcgggaggaacagtttagtaccacattgcccagctaactcgaacgcgaccaacttataaacccgcgcgctgtcgcttgtgtgGCCCCCCACCACCGCGCAAGgttttagagctatgctgaaaagcatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttt
在一种实现方式中，所采用的Cas9核酸酶表达框具体为(Seq ID No.2)：ctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaatgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcacggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgcagcccgggggatccccaatacttgtatggccgcggccgctctagatggattacaaggaccacgacggggattacaaggaccacgacattgattacaaggatgatgatgacaagatggctccgaagaagaagaggaaggttggcatccacggggtgccagctgctgacaagaagtactcgatcggcctcgatattgggactaactctgttggctgggccgtgatcaccgacgagtacaaggtgccctcaaagaagttcaaggtcctgggcaacaccgatcggcattccatcaagaagaatctcattggcgctctcctgttcgacagcggcgagacggctgaggctacgcggctcaagcgcaccgcccgcaggcggtacacgcgcaggaagaatcgcatctgctacctgcaggagattttctccaacgagatggcgaaggttgacgattctttcttccacaggctggaggagtcattcctcgtggaggaggataagaagcacgagcggcatccaatcttcggcaacattgtcgacgaggttgcctaccacgagaagtaccctacgatctaccatctgcggaagaagctcgtggactccacagataaggcggacctccgcctgatctacctcgctctggcccacatgattaagttcaggggccatttcctgatcgagggggatctcaacccggacaatagcgatgttgacaagctgttcatccagctcgtgcagacgtacaaccagctcttcgaggagaaccccattaatgcgtcaggcgtcgacgcgaaggctatcctgtccgctaggctctcgaagtctcggcgcctcgagaacctgatcgcccagctgccgggcgagaagaagaacggcctgttcgggaatctcattgcgctcagcctggggctcacgcccaacttcaagtcgaatttcgatctcgctgaggacgccaagctgcagctctccaaggacacatacgacgatgacctggataacctcctggcccagatcggcgatcagtacgcggacctgttcctcgctgccaagaatctgtcggacgccatcctcctgtctgatattctcagggtgaacaccgagattacgaaggctccgctctcagcctccatgatcaagcgctacgacgagcaccatcaggatctgaccctcctgaaggcgctggtcaggcagcagctccccgagaagtacaaggagatcttcttcgatcagtcgaagaacggctacgctgggtacattgacggcggggcctctcaggaggagttctacaagttcatcaagccgattctggagaagatggacggcacggaggagctgctggtgaagctcaatcgcgaggacctcctgaggaagcagcggacattcgataacggcagcatcccacaccagattcatctcggggagctgcacgctatcctgaggaggcaggaggacttctaccctttcctcaaggataaccgcgagaagatcgagaagattctgactttcaggatcccgtactacgtcggcccactcgctaggggcaactcccgcttcgcttggatgacccgcaagtcagaggagacgatcacgccgtggaacttcgaggaggtggtcgacaagggcgctagcgctcagtcgttcatcgagaggatgacgaatttcgacaagaacctgccaaatgagaaggtgctccctaagcactcgctcctgtacgagtacttcacagtctacaacgagctgactaaggtgaagtatgtgaccgagggcatgaggaagccggctttcctgtctggggagcagaagaaggccatcgtggacctcctgttcaagaccaaccggaaggtcacggttaagcagctcaaggaggactacttcaagaagattgagtgcttcgattcggtcgagatctctggcgttgaggaccgcttcaacgcctccctggggacctaccacgatctcctgaagatcattaaggataaggacttcctggacaacgaggagaatgaggatatcctcgaggacattgtgctgacactcactctgttcgaggaccgggagatgatcgaggagcgcctgaagacttacgcccatctcttcgatgacaaggtcatgaagcagctcaagaggaggaggtacaccggctgggggaggctgagcaggaagctcatcaacggcattcgggacaagcagtccgggaagacgatcctcgacttcctgaagagcgatggcttcgcgaaccgcaatttcatgcagctgattcacgatgacagcctcacattcaaggaggatatccagaaggctcaggtgagcggccagggggactcgctgcacgagcatatcgcgaacctcgctggctcgccagctatcaagaaggggattctgcagaccgtgaaggttgtggacgagctggtgaaggtcatgggcaggcacaagcctgagaacatcgtcattgagatggcccgggagaatcagaccacgcagaagggccagaagaactcacgcgagaggatgaagaggatcgaggagggcattaaggagctggggtcccagatcctcaaggagcacccggtggagaacacgcagctgcagaatgagaagctctacctgtactacctccagaatggccgcgatatgtatgtggaccaggagctggatattaacaggctcagcgattacgacgtcgatcatatcgttccacagtcattcctgaaggatgactccattgacaacaaggtcctcaccaggtcggacaagaaccggggcaagtctgataatgttccttcagaggaggtcgttaagaagatgaagaactactggcgccagctcctgaatgccaagctgatcacgcagcggaagttcgataacctcacaaaggctgagaggggcgggctctctgagctggacaaggcgggcttcatcaagaggcagctggtcgagacacggcagatcactaagcacgttgcgcagattctcgactcacggatgaacactaagtacgatgagaatgacaag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附图说明
图1为通过PEG介导原生质体瞬时转化再生水稻株系中Ehd3基因定点突变测序检测的部分结果图，其中WT表示为野生型基因，“-”表示发生了删除突变的序列，“+”表示发生了插入突变的序列，“-/+”后边的数字表示删除或插入的核苷酸数量，阴影部分表示目标靶位。
具体实施方式
下述实施例中所使用的试验方法如无特殊说明，均为常规方法。下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下面介绍本发明一个实施例所采用的育种方法。
一、用于水稻Ehd3基因打靶的重组载体的制备。
1.1，选择水稻Ehd3基因(LOC_Os08g01420)第一外显子上中自翻译起始密码子ATG后第17-39位的核苷酸序列GCCCCCCACCACCGCGCAAGAGG，(下划线部分为所述5’-(N)X-NGG-3’结构中NGG部分)，作为打靶位点。
1.2，按所选择靶位点合成(华大基因公司)正向寡核苷酸链(Ehd3KO1P1)和可与之互补的反向寡核苷酸链(Ehd3KO1P2)，
具体序列为：
Ehd3KO1P1:TGTGGCCCCCCACCACCGCGCAAG；
Ehd3KO1P2:AAACCTTGCGCGGTGGTGGGGGGC。
其中未被下划线标注的部分为上述靶位点中去除NGG的序列或互补序列，下划线部分为用于连接载体的粘性末端。
1.3,经过退火程序，将Ehd3KO1P1和Ehd3KO1P2两链退火形成具有粘性末端的双链DNA，作为构建重组载体的插入片段。
1.4，用BsaI内切酶(NEB公司)在37℃酶切包含能够在水稻细胞内表达的向导RNA表达框(核苷酸序列如Seq ID No.1所示)和能够在水稻细胞内表达的Cas9核酸酶表达框的水稻CRISPR/Cas9基因工程载体(核苷酸序列如Seq ID No.2所示)，载体结构和构建方法按文献所示(Xuet al,Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cassystem in rice,RICE,2014)所示，使用BsaI内切酶酶切水稻CRISPR/Cas9基因工程载体2小时，65℃失活酶切体系10分钟，作为构建重组载体的骨架片段。
1.5，用T4连接酶(NEB公司)将重组载体骨架片段和插入片段相连，转入大肠杆菌中。经测序验证后，提取阳性转化子，构成用于水稻Ehd32基因CRISPR/Cas9打靶的重组载体质粒。
二、原生质体瞬时转化介导的水稻Ehd3基因打靶和生育期的改良。
2.1,利用PEG法将所述用于水稻Ehd3基因CRISPR/Cas9打靶的重组载体质粒转化至杂交水稻不育系培矮64S原生质体，水稻原生质体转化具体过程参考了文献Zhang等A highly efficient rice green tissue protoplastsystem for transient gene expression and studying light/chloroplast-relatedprocesses.Plant Method(2011).中公开的实验方法。
2.2，利用所述转化后的培矮64S水稻原生质体，获得再生水稻植株，共9株。水稻原生质体转化和植株再生具体过程参考了文献Hayashimoto等A Polyethylene Glycol-Mediated Protoplast Transformation System forProduction of Fertile Transgenic Rice Plants.Plant Physiology(1990).中公开的实验方法。
2.3，利用植物基因组小量提取试剂盒(天根生化公司)，提取所获18棵再生植株的基因组DNA。以该DNA为模板，用Phusion高保真DNA聚合酶(NEB公司)PCR扩增包含靶标区域的序列，其中PCR扩增所用的引物为：
Ehd3KO1genome check FP:GCTCCGTCCGTCGTCTCCTCCC
Ehd3KO1genome check RP:GGAGGAAGCCGTGGAGAGTGTC
2.4，以Ehd3KO1genome check FP为引物对所获PCR扩增片段直接测序，分析靶标位点的突变。测序结果表明，在所测9棵植株中，4棵植株带有Ehd3基因靶标序列上的突变，突变效率为44.4％；突变的形式包括碱基的插入和/或缺失。部分结果如图1所示；其中两个等位Ehd3基因都出现功能缺失突变的再生株系为2株，同时出现等位基因效率为22.2％。图1为通过PEG介导原生质体瞬时转化再生水稻株系中Ehd3基因定点突变测序检测的部分结果图。图中阴影部分，即第7位开始的GCCCCCCACCACCGCGC-AAG为打靶的目标位。
2.5，观察两个等位Ehd3基因都出现功能缺失突变的再生株系及自交产生子一代的生育期，所有2株系子代的生育期均为129天～131天左右，较供体品种培矮64S的生育期114天，均推迟2周以上，表明所述2株系的生育期被成功推迟。
相对于传统育种方法，本方法有以下优点
①，育种周期短，整个材料定向创制过程可在7个月内完成，而传统杂交-回交方法至少需要3～5年时间。
②，只改变了受体品种的一个基因，所获材料生育期延长之外其他农艺性状不变，而传统回交方法会导入与Ehd3连锁的其他基因，可能影响受体品种的农艺性状。
Claims (8)
1.一种延长水稻生育期的育种方法，其特征在于，所述方法包括如下步骤：
步骤1，在水稻生育期决定基因Ehd3外显子区选取靶标片段，所述靶标片段的双链结构的一条链具有5’-(N)X-NGG-3’结构，(N)X代表数目为X的一段碱基序列{N1,N2……Nx}，X代表碱基的数量，N1,N2……Nx中的每一个表示A、G、C、T中的任意一个，NGG中的N也表示A、T、G、C中的一种；
步骤2，利用所述靶标片段，构建用于水稻Ehd3基因打靶的CRISPR/Cas9重组载体，其中所述重组载体中包含具有所述靶标片段的向导RNA表达框，以及Cas9核酸酶表达框；
步骤3，将所述重组载体导入水稻细胞，诱导所述向导RNA表达框和所述Cas9核酸酶表达框在水稻细胞中共同表达；
步骤4，剪切Ehd3基因的双链靶标片段，诱发水稻细胞自身的DNA修复功能，在靶标位点随机插入或缺失碱基，实现细胞内Ehd3基因的功能缺失突变；
步骤5，用导入所述重组载体的水稻细胞再生水稻植株；
步骤6，对再生植株中Ehd3基因包含靶标片段的DNA区段测序。
步骤7，选择两个等位Ehd3基因都出现功能缺失突变的再生株系，进行表型鉴定，观察再生株系子代的生育期，挑取生育期推迟超过预定时间段的株系，作为所创制的生育期推迟的水稻材料。
2.根据权利要求1所述的育种方法，其特征在于，X为19或20。
3.根据权利要求1所述的育种方法，其特征在于，所述向导RNA表达框和所述Cas9核酸酶表达框均能够在水稻细胞中表达。
4.根据权利要求3所述的育种方法，其特征在于，所述向导RNA表 达框的核苷酸序列如Seq ID No.1所示；所述Cas9核酸酶表达框的核苷酸序列如Seq ID No.2所示。
5.根据权利要求3所述的育种方法，其特征在于，
所述向导RNA表达框包括：水稻U6启动子，其核苷酸序列如Seq ID No.1第1至246位所示；结构特征为(N)X的靶标序列和人工合成的sgRNA骨架序列，其核苷酸序列如Seq ID No.1第267至350位所示；以及Poly-T终止子，其核苷酸序列如Seq ID No.1第351至358位所示。
6.根据权利要求3所述的育种方法，其特征在于，
所述Cas9核酸酶表达框包括：玉米ZmUBI启动子，其核苷酸序列如Seq ID No.2第1至2031位所示；植物偏好密码子改造后的Cas9编码序列，其核苷酸序列如Seq ID No.2第2034至6305位所示；以及tNOS终止子，其核苷酸序列如Seq ID No.2第6347至6599位所示。
7.根据权利要求1所述的育种方法，其特征在于，在所述步骤7中，挑取子代生育期推迟超过两周的株系作为所创制的生育期推迟的水稻材料。
8.根据权利要求1所述的育种方法，其特征在于，所述步骤7还包括对所得植株进行自交，观察再生株系子代的生育期。
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