利用CRISPR/Cas-9技术创制水稻温敏核不育系

doi:10.19303/j.issn.1008-0384.2018.10.001

福建农业学报 ›› 2018, Vol. 33 ›› Issue (10): 1011-1015.doi: 10.19303/j.issn.1008-0384.2018.10.001

• 作物科学 •

利用CRISPR/Cas-9技术创制水稻温敏核不育系

吴明基, 林艳, 刘华清, 陈建民, 付艳萍, 杨绍华, 王锋

福建省农业科学院生物技术研究所, 福建福州 350003

收稿日期:2018-08-16 修回日期:2018-09-29 发布日期:2019-03-28
通讯作者: 王锋(1963-),男,博士,研究员,研究方向:水稻基因工程与遗传育种(E-mail:wf@fjage.org) E-mail:wf@fjage.org
作者简介:吴明基(1976-)男,助理研究员,研究方向:水稻遗传育种(E-mail:839845032@qq.com)
基金资助:
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）

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

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

Received:2018-08-16 Revised:2018-09-29 Published:2019-03-28

摘要/Abstract

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。

关键词: 杂交水稻, 两系不育系, 基因, CRISPR/Cas9, tms5

Abstract: 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 T₀ 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 T₁ generation of the non-transgenic plants. The mutants showed the same characteristics as shown in the T₀ generation without any new mutations. Male fertility of T₂ 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.

Key words: hybrid rice, two-line male sterile line, gene, CRISPR/Cas-9, tms5

中图分类号:

吴明基, 林艳, 刘华清, 陈建民, 付艳萍, 杨绍华, 王锋. 利用CRISPR/Cas-9技术创制水稻温敏核不育系[J]. 福建农业学报, 2018, 33(10): 1011-1015.

WU Ming-ji, LIN Yan, LIU Hua-qing, CHEN Jian-min, FU Yan-ping, YANG Shao-hua, WANG Feng. Development of Thermo-Sensitive Male Sterile Rice with CRISPR/Cas9 Technology[J]. Fujian Journal of Agricultural Sciences, 2018, 33(10): 1011-1015.

参考文献

[1] 朱英国. 杂交水稻研究50年[J]. 科学通报, 2016, 61(35):3740-3747.
[2] 余四斌, 熊银, 肖景华, 等. 杂交稻与绿色超级稻[J]. 科学通报,2016,61(35):3797-3803.
[3] 牟同敏. 中国两系法杂交水稻研究进展和展望[J]. 科学通报, 2016, 61(35):3761-3769.
[4] 范优荣, 曹晓风, 张启发. 光温敏雄性不育水稻的研究进展[J].科学通报,2016,61(35):3822-3832.
[5] FAN Y, YANG J, MATHIONI S M, et al. PMS1T, producing phased small-interfering RNAs, regulates photoperiod-sensitive male sterility in rice[J].Proceedings of the National Academy of Sciences, 2016, 113(52):15144-15149.
[6] DING J, LU Q, OUYANG Y, et al. A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice[J]. Proceedings of the National Academy of Sciences, 2012, 109(7):2654-2659.
[7] ZHOU H, ZHOU M, YANG Y, et al. RNase Z^S1 processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice[J]. Nature Communications, 2014(5):4884.
[8] ZHANG H, XU C, He Y, et al. Mutation in CSA creates a new photoperiod-sensitive genic male sterile line applicable for hybrid rice seed production[J]. Proceedings of the National Academy of Sciences, 2013, 110(1):76-81.
[9] YU J, HAN J, KIM Y, et al. Two rice receptor-like kinases maintain male fertility under changing temperatures[J]. Proceedings of the National Academy of Sciences, 2017, 114(46):12327-12332.
[10] ZHANG J, ZHANG H, BOTELLA J R, et al. Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties[J]. Journal of Integrative Plant Biology, 2018, 60:369-375.
[11] WANG F, WANG C, LIU P, et al. Enhanced rice blast resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF transcription factor gene OsERF922[J]. PLoS ONE, 2016(1):e0154027.
[12] TANG L, MAO B, LI Y, et al. Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield[J]. Scientific Reports, 2017(7):1-12.
[13] SUN Y, ZHANG X, WU C, et al. Engineering herbicide resistant rice plants through CRISPR/Cas9-Mediated homologous recombination of acetolactate synthase[J]. Molecular Plant, 2016(9):628-631.
[14] MACOVEI A, SEVILLA NR, CANTOS C, et al. Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to Rice tungro spherical virus[J]. Plant Biotechnology Journal, 2018,1-10.doi:10.1111/pbi.12927.
[15] LI Q, ZHANG D, CHEN M, et al. Development of japonica photo-sensitive genic male sterile rice lines by editing carbon starved anther using CRISPR/Cas9[J]. Journal of Genettics Genomics, 2016,43:415-419.
[16] ZHOU H, HE M, LI J, et al. Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system[J]. Scientific Reports, 2016(6):1-12.
[17] HIEI Y, KOMARI T, KUBO T. Transformation of rice mediated by Agrobacterium tumefaciens[J]. Plant Molecular Biology, 1997, 35:205-218.

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

HTML

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	姚锦爱, 张鸿, 黄鹏, 陈峰, 余德亿. 建兰茎腐病原菌尖孢镰刀菌F-02的绿色荧光蛋白基因标记[J]. 福建农业学报, 2019, 34(1): 70-75.
[2]	张柱坚, 陈子强, 顾建强, 田大刚. 稻瘟病抗性基因Pi-d2、Pi-d3和Pigm不同敲除突变体的抗性评价[J]. 福建农业学报, 2018, 33(12): 1231-1236.
[3]	高晨, 郑玉成, 周珍, 叶卓昕, 陈丹, 孙云, 叶乃兴. 茶树乙醇脱氢酶基因CsADH2的克隆与表达分析[J]. 福建农业学报, 2018, 33(12): 1257-1263.
[4]	辛清武, 刘艺展, 朱志明, 李丽, 章琳俐, 缪中纬, 郑嫩珠. 连城白鸭MITF基因的cDNA克隆、组织表达与分析[J]. 福建农业学报, 2018, 33(12): 1292-1300.
[5]	田容川, 黄薇. 鸡肠道大肠杆菌多抗菌株C20耐药性基因的预测和分析[J]. 福建农业学报, 2017, 32(9): 932-938.
[6]	傅秋玲, 傅光华, 陈翠腾, 程龙飞, 万春和, 施少华, 陈红梅, 陈珍, 朱春华, 黄瑜. 半番鸭源鸭1型甲肝病毒亚型的分离鉴定及其VP1基因分析[J]. 福建农业学报, 2017, 32(8): 813-817.
[7]	郑家团, 周鹏, 涂诗航, 刘庸庆, 邓则勤. 感光型杂交水稻新品种元优2105的选育[J]. 福建农业学报, 2017, 32(5): 491-494.
[8]	谢婉凤, 李慧敏, 黄爱珍, 冯丽贞, 张飞萍. 马尾松响应松材线虫侵染的基因动态表达变化[J]. 福建农业学报, 2017, 32(4): 403-409.
[9]	芮祥云, 陈金芝, 黄鹏榕, 杨广. DNA干扰现象及其作用机理研究进展[J]. 福建农业学报, 2017, 32(4): 456-460.
[10]	林能锋, 陈强, 杨金先, 许斌福, 曾红. 鳗鲡幼苗种类的DNA指纹鉴定[J]. 福建农业学报, 2017, 32(3): 258-262.
[11]	卢荣辉, 刘小龙, 肖隆华, 刘荣昌, 傅光华, 苏敬良, 祁保民, 黄瑜. 鸭甲型病毒性肝炎最新研究进展[J]. 福建农业学报, 2017, 32(3): 336-341.
[12]	万春和, 程龙飞, 陈红梅, 傅光华, 傅秋玲, 施少华, 陈翠腾, 刘荣昌, 黄瑜. 鸭疫里默氏菌铁依赖抑制子基因的原核表达[J]. 福建农业学报, 2017, 32(2): 107-110.
[13]	尹亚军, 张涛, 王令, 路宏朝, 杜伟立. 汉中黑稻花青素合成酶基因克隆及生物信息学分析[J]. 福建农业学报, 2017, 32(2): 124-129.
[14]	陈婷, 周平, 刘鑫铭, 蔡盛华, 雷龑. 基于高通量测序的福安刺葡萄基因组初步研究[J]. 福建农业学报, 2017, 32(2): 134-137.
[15]	许惠滨, 魏毅东, 谢鸿光, 吴方喜, 罗曦, 朱永生, 江敏榕, 蒋家焕, 林强, 连玲, 蔡秋华, 潘丽燕, 谢华安, 张建福. 耐储藏籼型杂交水稻恢复系福恢7185的创制与利用[J]. 福建农业学报, 2017, 32(12): 1275-1280.