利用CRISPR/Cas-9技术创制水稻温敏核不育系  

Development of Thermo-Sensitive Male Sterile Rice with CRISPR/Cas9 Technology

作　　者：吴明基[1] 林艳[1] 刘华清[1] 陈建民[1] 付艳萍[1] 杨绍华[1] 王锋[1] WU Ming-ji;LIN Yan;LIU Hua-qing;CHEN Jian-min;FU Yan-ping;YANG Shao-hua;WANG Feng(Institute of Biotechnology,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 350003,China)

机构地区：[1]福建省农业科学院生物技术研究所,福建福州350003

出　　处：《福建农业学报》2018年第10期1011-1015,共5页Fujian Journal of Agricultural Sciences

基　　金：福建省自然科学基金项目(2016J01132);福建省科技计划项目--省属公益类科研院所基本科研专项(2016R1018-11);福建省科技计划重大专项(2015NZ0002-3)

摘　　要：水稻光温敏核不育系的选育是两系法杂交稻选育的关键环节。温敏不育基因tms5在生产上应用最为广泛,通过CRISPR/Cas9基因组编辑技术突变可育水稻品种的TMS5基因,可以快速选育水稻温敏型两系不育系。利用CRISPR/Cas-9技术创制水稻温敏核不育系的试验结果表明:通过构建CRISPR/Cas-9基因编辑载体TMS502,转化优良中间材料GH89获得10株T0代转基因苗,有6株产生了插入或缺失突变,其中纯合突变株tms5-1和双等位突变株tms5-4在T0代就表现出温敏不育特征;T1代非转基因单株中共有4种纯合突变基因型,突变类型与T0代检测结果一致,并未产生新的突变;T2代tms5不育系材料的温敏不育起点温度约为24℃,符合水稻两系不育系选育要求。研究结果证明了利用基因编辑技术培育水稻两系不育系的有效性,所创制的不育系可供进一步选育利用。Breeding of photoperiod or thermo-sensitive male sterile line is the key for the development of two-line hybrid rice.The allele of tms5 is the major TGMS genetic resource for two-line hybrid rice breeding in China.Targeted editing on TMS5 of fertile rice using CRISPR/Cas-9 technology could effectively create thermo-sensitive genic male sterile rice.This study used the approach to edit the TMS5 gene successfully.The CRISPR/Cas-9 vector of TMS502 was constructed,then the T-DNA was transformed into GH89.Ten transgenic plants were thus regenerated,with 6 of them containing either the insertion or deletion mutations.The T 0 plant of tms5-l was a homozygous mutant,and tms 5-4 a bi-allelic mutant.Both of them displayed thermo-sensitive male sterile phenotype.There were 4 homozygous mutant genotypes in the T 1 generation of the non-transgenic plants.The mutants showed the same characteristics as shown in the T 0 generation without any new mutations.Male fertility of T 2 generation of tms5 mutants was tested in natural high temperature environment and in phytotrons at 3 temperature levels.The results indicated that the critical sterility-inducing temperature of the tms5 sterile lines was approximately 24 C,which satisfied the requirements for two-line male sterile line breeding.The successful conversion of GH89 line into a two-line male sterile rice using the gene editing technology suggested that CRISPR/Cas-9 could be applied to facilitate the development of two line hybrid rice breeding in the future.

关 键 词：杂交水稻 两系不育系 基因 CRISPR/Cas9 tms5 

分 类 号：Q81[生物学—生物工程] S511[农业科学—作物学]