CN113322266B - 水稻OsRHD1-1基因在水稻雄性核不育系培育中的应用 - Google Patents
水稻OsRHD1-1基因在水稻雄性核不育系培育中的应用 Download PDFInfo
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Abstract
本发明公开了水稻OsRHD1‑1基因在水稻雄性核不育系培育中的应用。本发明通过设计OsRHD1‑1基因的敲除靶点构建基因敲除载体，并在水稻中进行遗传转化，发现OsRHD1‑1基因功能的丧失能够导致花粉不育，有助于新型雄性核不育系的培育，助力杂交育种的发展。
Description
技术领域
本发明涉及生物工程技术领域，特别是涉及水稻OsRHD1-1基因在水稻雄性核不育系培育中的应用。
背景技术
水稻是世界上最重要的粮食作物之一，怎样提高水稻产量来满足日益增长的粮食需求是当今需要迫切解决的问题。由于我国耕地面积和淡水资源有限，提高水稻单产的方法是提高水稻产量的主要途径。影响水稻单产的因素有很多，影响水稻每株分蘖数、每穗粒数和粒重的内在遗传特性是水稻单产决定因素；外在因素，包括光照、温度、水分和盐度等非生物胁迫以及病害和虫害的生物胁迫对单产有着重要的影响；另外，人为因素也对水稻单产起着关键的作用，比如栽培方式、施肥方式以及病虫害防治水平等。
水稻的高产离不开优良的品种，优良品种的选育离不开有效的育种方法。当前，水稻育种方法包括常规育种和分子育种。常规育种包括***育种，杂交育种和诱变育种；分子育种包括分子标记辅助选择育种，转基因育种和分子设计育种。但是由于分子育种目前还存在成本高，安全担忧以及水稻主要农艺性状基因的发掘和功能研究不透彻等问题，其应用仍旧受很大的限制。目前，***育种仍旧是主要的育种方法，而其中的杂交育种应用极为广泛，大大提高了我国水稻的产量。
杂交育种就是利用具有遗传差异的两个水稻亚种进行杂交，选择的两个亚种的优良性状能够互补，且后代能够产生杂种优势的育种方法。目前杂交育种方法有三系法和两系法。三系法包括不育系(核质互作雄性不育系和雄性核不育系)、保持系和恢复系；两系法包括不育系(温敏雄性核不育系和光敏雄性核不育系)和恢复系。目前我国使用的三系法不育系主要是II-32A、金23A、珍汕97A、冈46A和龙特浦A；两系法不育系主要是Y58S和培矮64S。因此，不育系的培育对杂交水稻产业的发展具有关键的作用。然而，不育系品质低劣是造成杂交水稻产量品质不好的重要原因，因此，培育米质好、抗逆性强以及配合力好的不育系是当前急需解决的问题。
在拟南芥中，Schiefelbein等(J.W.Schiefelbein andC.Somerville.1990.Genetic Control of Root Hair Development in Arabidopsisthaliana.Plant Cell,2(3):235-243.)报道了拟南芥ROOT HAIR DEFECTIVE 1(AtRHD1，At1G64440)基因在根毛的发育过程中具有重要的调控作用，Atrhd1突变体表现为根毛起始异常。
通过序列比对分析发现，水稻中存在4个与AtRHD1功能相似的同源基因，编码UDP-葡萄糖/半乳糖差向异构酶，但还没有这些基因调控水稻花粉发育的功能的报道。
发明内容
本申请研究结果表明OsRHD1-1基因(LOC-Os05g51670)在花粉发育中，起着关键的作用。由于OsRHD1-1是一个细胞核基因，在水稻中敲除该基因能够导致花粉败育，因而可以作为培育新型水稻雄性核不育系的育种材料，可以为水稻杂交育种提供优良品种的杂交组合，进而提高水稻的品质和产量。
水稻OsRHD1-1基因在水稻雄性核不育系培育中的应用。优选的，水稻OsRHD1-1基因的碱基序列如SEQ ID NO.1所示。
本发明又提供了一种水稻雄性核不育系培育方法，将水稻OsRHD1-1基因敲除。
优选的，所述的水稻雄性核不育系培育方法，包括以下步骤：
(1)构建基因敲除载体，所述基因敲除载体为带有用于对水稻OsRHD1-1基因进行敲除的序列的植物表达载体；
(2)将步骤(1)基因敲除载体导入水稻的细胞中将水稻OsRHD1-1基因敲除，培养后获得水稻雄性核不育系。
优选的，所述植物表达载体为pYLCRISPR/Cas9Pubi-H。
转化受体植物时，可采用农杆菌介导转化的方法，所述的农杆菌具体可以为农杆菌EHA105。步骤(2)将基因敲除载体转入农杆菌后，侵染水稻。
优选的，农杆菌侵染的水稻为水稻种子诱导的愈伤组织。除了水稻种子诱导的愈伤组织，也可以是水稻植株上取的组织样品诱导的愈伤组织。或者其他在转基因后能够培育出植株的方式都可以使用。
优选的，基因敲除针对的靶点碱基序列为5’-tcctccagcttctccaact-3’。基因敲除的靶点碱基序列不局限于该序列，只要能够使得水稻的OsRHD1-1基因被敲除后基因功能丧失即可。
本发明具备的有益效果：本发明通过设计OsRHD1-1基因的敲除靶点构建基因敲除载体，并在水稻中进行遗传转化，发现OsRHD1-1基因功能的丧失能够导致花粉不育，有助于新型雄性核不育系的培育，助力杂交育种的发展。
附图说明
图1为Osrhd1-1-1、Osrhd1-1-2、Osrhd1-1-3敲除株系的OsRHD1-1基因组序列编辑情况检测结果图。
图2为野生型(NIP)和水稻敲除株系(Osrhd1-1-1、Osrhd1-1-2、Osrhd1-1-3)的小花对比结果图。
图3为野生型(NIP)和水稻敲除株系(Osrhd1-1-1、Osrhd1-1-2、Osrhd1-1-3)的花粉育性和花药对比结果图。
图4为野生型(NIP)和水稻敲除株系(Osrhd1-1-1、Osrhd1-1-2、Osrhd1-1-3)的雌蕊形态对比结果图。
具体实施方式
实施例1：水稻OsRHD1-1基因敲除载体构建和水稻遗传转化
水稻OsRHD1-1基因的碱基序列如SEQ ID NO.1所示。
(1)根据“http://skl.scau.edu.cn/targetdesign/”网站，设计OsRHD1-1基因的特异性敲除靶点，序列如下：
敲除靶点：TCCTCCAGCTTCTCCAACT。
根据敲除靶点序列设计表达对应sgRNA序列的表达序列。
(2)利用NEB公司的BsaI内切酶双酶切(同一种酶切两个位置)pYLCRISPR/Cas9Pubi-H载体
体系如下：
酶切条件为：37℃30分钟。
(3)敲除靶点合成
合成的引物靶点序列如下：
595100-aFP：5’-GCCGTCCTCCAGCTTCTCCAACT-3’；
595100-aRP：5’-AAACAGTTGGAGAAGCTGGAGGA-3’。
引物退火体系如下：
595100-aFP：9μL；595100-aRP：9μL；ddH2O加至总体积20μL。
退火条件为：95℃5分钟，之后取出室温放置。
(4)敲除靶点与酶切载体的连接
使用Taraka公司的T4连接酶进行连接反应，体系如下：
敲除靶点：1μL；酶切载体：1.5μL；T4连接酶缓冲液(T4 ligase buffer)：0.5μL；T4连接酶(T4 ligase)：0.5μL；ddH2O加至总体积5μL。
连接反应条件为：10℃3分钟，6℃6秒(每循环加0.2℃)，16℃3分钟，18℃1分钟，第一步至第四步19个循环；65℃15分钟；12℃保存。
取连接产物转化大肠杆菌DH5α，挑取PCR验证阳性克隆后测序验证，并提取质粒备用。
(5)从-80℃冰箱中取出EHA105感受态细胞，利用液氮冻融法转化农杆菌。
感受态细胞置于冰上解冻，取5μL目的质粒加到感受态细胞中，混匀后置于冰上30min左右，再将离心管内的所有混合物放至液氮中速冻1分钟，之后放37℃水浴锅2分钟融化，之后加入1mL的无抗LB液体营养基，重悬细胞，放置28℃200rpm摇床上复苏2小时，之后涂布于含有相应抗性的YEP平板上(Rif(利福平)50mg/L，Kan(硫酸卡那霉素)50mg/L)，28℃倒置培养2天以至长出单菌落。
(7)挑取长出的农杆菌单克隆，菌落PCR鉴定阳性单克隆，
(8)用水稻快速遗传转化法转化水稻种子(日本晴水稻)诱导的愈伤。
(9)潮霉素筛选抗性愈伤并诱导分化出苗，提取分化苗叶片DNA，扩增基因组目标序列测序鉴定靶点突变情况，所用引物如下：
RHD1-1CAS-F：5’-GAAGCTAGGAAAAAGGGAAATA-3’；
RHD1-1CAS-R：5’-TCGCCCGAATACAGATATAAGT-3’。
实施例2：水稻OsRHD1-1基因敲除株系基因编辑情况
为了检测水稻OsRHD1-1基因敲除株系小花中花粉发育情况，提取基因编辑苗的DNA，扩增片段送杭州尚亚公司测序。
如图1所示，测序结果发现，测序的3个株系的两条DNA单链均发生了变异。
实施例3：水稻OsRHD1-1基因敲除株系小花花药性状观察
水稻OsRHD1-1基因敲除株系小花中花药变小，颜色变浅，发育不良。
对实施例2获得的水稻OsRHD1-1基因敲除株系小花性状观察，如图2所示，在水稻OsRHD1-1基因敲除株系小花中花药变小，颜色变浅，发育不良。
实施例4：水稻OsRHD1-1基因敲除株系花粉观察
为了检测水稻OsRHD1-1基因敲除株系小花中花粉发育情况，使用碘染法对花粉进行了观察。
结果如图3所示，水稻OsRHD1-1基因敲除株系小花中无花粉，为花粉不育。
实施例5：水稻OsRHD1-1基因敲除株系小花雌蕊观察
为了检测水稻OsRHD1-1基因敲除株系小花中雌蕊发育情况，使用碘染法对雌蕊形态进行了观察。
结果如图4所示，水稻OsRHD1-1基因敲除株系小花雌蕊发育正常。
我们的实验成果以日本晴水稻背景，证明了水稻OsRHD1-1基因在花粉的发育中具有关键作用，其功能的丧失导致无花粉表型，且不影响雌蕊的生长。
序列表
<110> 浙江大学
<120> 水稻OsRHD1-1 基因在水稻雄性核不育系培育中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1065
<212> DNA
<213> 水稻(Oryza sativa L.)
<400> 1
atggtttcgg ccttgttgcg gacgatcctg gtgacgggcg gcgccggcta catcggcagc 60
cacaccgtcc tccagcttct ccaactcggc ttccgcgttg tcgtcctcga caacctcgac 120
aacgcctccg agctcgccat cctccgcgtc agggaactcg ccggacacaa cgccaacaac 180
ctcgacttcc gcaaggttga cctccgcgac aagcaagcgt tggaccaaat cttctcctct 240
caaaggtttg aggctgtcat ccattttgcc gggctgaaag ctgttggcga gagcgtgcag 300
aagcccctgc tttactacga caacaacctc atcggcacca tcactctcct gcaggtcatg 360
gccgcacatg gctgcaccaa gctggtgttc tcatcatccg caactgtcta cgggtggccc 420
aaggaggtgc cctgcactga agaatcccca ctttgtgcaa tgaaccccta cggcagaaca 480
aagctggtaa tcgaagacat gtgccgggat ctgcatgcct cagacccaaa ctggaagatc 540
atactgctcc gatacttcaa ccctgttgga gctcacccaa gcgggtacat tggtgaggac 600
ccctgcggca tcccaaacaa cctcatgccc ttcgtccagc aggtcgctgt tggcaggagg 660
ccggccctta ccgtctatgg aaccgactac aacaccaagg atggaactgg ggttcgtgac 720
tatatccatg ttgttgatct agcggatggt catatcgccg cgttaaggaa gctctatgaa 780
gattctgata gaataggatg tgaggtgtac aatctgggca ctggaaaggg gacatctgtg 840
ctggaaatgg ttgcagcatt cgagaaagct tctggaaaga aaatcccgct tgtatttgct 900
ggacgaaggc ctggagatgc cgagatcgtt tacgctcaaa ctgccaaagc tgagaaggaa 960
ctgaaatgga aggcaaaata cggggtagag gagatgtgca gggacctgtg gaattgggcg 1020
agcaagaacc cctacgggta tggatcgccg gacagtagca actga 1065
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tcctccagct tctccaact 19
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gccgtcctcc agcttctcca act 23
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aaacagttgg agaagctgga gga 23
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaagctagga aaaagggaaa ta 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tcgcccgaat acagatataa gt 22
Claims (7)
1.水稻OsRHD1-1基因在水稻雄性核不育系培育中的应用，水稻OsRHD1-1基因的碱基序列如SEQ ID NO.1所示。
2.一种水稻雄性核不育系培育方法，其特征在于，将水稻OsRHD1-1基因敲除，水稻OsRHD1-1基因的碱基序列如SEQ ID NO.1所示。
3.如权利要求2所述的水稻雄性核不育系培育方法，其特征在于，包括以下步骤：
（1）构建基因敲除载体，所述基因敲除载体为带有用于对水稻OsRHD1-1基因进行敲除的序列的植物表达载体；
（2）将步骤（1）基因敲除载体导入水稻的细胞中将水稻OsRHD1-1基因敲除，培养后获得水稻雄性核不育系。
4.如权利要求3所述的水稻雄性核不育系培育方法，其特征在于，所述植物表达载体为pYLCRISPR/Cas9Pubi-H。
5.如权利要求3所述的水稻雄性核不育系培育方法，其特征在于，步骤（2）将基因敲除载体转入农杆菌后，侵染水稻。
6.如权利要求5所述的水稻雄性核不育系培育方法，其特征在于，农杆菌侵染的水稻为水稻种子诱导的愈伤组织。
7.如权利要求3所述的水稻雄性核不育系培育方法，其特征在于，基因敲除针对的靶点碱基序列为5’-tcctccagcttctccaact-3’。
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| CN106011167A (zh) * | 2016-05-27 | 2016-10-12 | 上海交通大学 | 雄性不育基因OsDPW2的应用及水稻育性恢复的方法 |
| CN110241126A (zh) * | 2019-07-03 | 2019-09-17 | 浙江大学 | OsDGD2β基因在培育雄性不育水稻品种中的应用 |
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| CN106011167A (zh) * | 2016-05-27 | 2016-10-12 | 上海交通大学 | 雄性不育基因OsDPW2的应用及水稻育性恢复的方法 |
| CN110241126A (zh) * | 2019-07-03 | 2019-09-17 | 浙江大学 | OsDGD2β基因在培育雄性不育水稻品种中的应用 |
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| Title |
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| GenBank：AB087745.1;佚名;《NCBI》;20080215;参见序列 * |
| NCBI Reference Sequence: XM_ 015784747.2;佚名;《NCBI》;20180807;参见序列 * |
| UDP-glucose pyrophosphorylase gene affects mycelia growth and polysaccharide synthesis of Grifola frondosa;Xin-Yi Zan;《International Journal of Biological Macromolecules》;20200607;第1161-1170页 * |
| 水稻OsYAB6和OsUGE1基因克隆与功能分析;刘慧丽;《中国科学院植物研究所博士学位论文》;20110525;全文 * |
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