敲除TMS5基因获得温敏不育粳稻新材料

doi:10.16819/j.1001-7216.2019.9035

中国水稻科学 ›› 2019, Vol. 33 ›› Issue (5): 429-435.DOI: 10.16819/j.1001-7216.2019.9035

敲除TMS5基因获得温敏不育粳稻新材料

杜茜^1,², 费云燕^1,², 王芳权^1,², 许扬^1,², 王军^1,², 李文奇^1,², 赵凌^1,², 陈智慧^1,², 梁国华^1,², 周勇^1,², 杨杰^1,^2,^*()

¹扬州大学江苏省粮食作物现代产业技术协同创新中心, 江苏扬州 225009
²江苏省农业科学院粮食作物研究所 / 国家水稻改良中心南京分中心/ 江苏省优质水稻工程技术研究中心, 南京 210014

收稿日期:2019-03-28 修回日期:2019-04-23 出版日期:2019-09-10 发布日期:2019-09-10
通讯作者: 杨杰
基金资助:
国家重点研发计划资助项目(2017YFD0100403);江苏省农业科技自主创新资金资助项目[CX(18)2022];江苏省现代农业重点研发计划资助项目(BE2017345);国家转基因生物新品种培育科技重大专项(2018ZX0800102);江苏省自然科学基金资助项目(BK20170610)

Thermo-sensitive Male Sterile Line Created by Editing TMS5 Gene in japonica Rice

Xi DU^1,², Yunyan FEI^1,², Fangquan WANG^1,², Yang XU^1,², Jun WANG^1,², Wenqi LI^1,², Ling ZHAO^1,², Zhihui CHEN^1,², Guohua LIANG^1,², Yong ZHOU^1,², Jie YANG^1,^2,^*()

¹ Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China
² Institute of Food Crops, Jiangsu Academy of Agricultural Sciences / Nanjing Branch of Chinese National Center for Rice Improvement / Jiangsu High Quality Rice R & D Center, Nanjing 210014, China;

Received:2019-03-28 Revised:2019-04-23 Online:2019-09-10 Published:2019-09-10
Contact: Jie YANG

摘要/Abstract

摘要：

【目的】 水稻温敏不育系的选育是两系杂交稻育种工作中的关键环节。TMS5是控制温敏不育的重要基因,在生产上也应用较广。为了加快水稻温敏不育系的选育进程,【方法】 利用CRISPR/Cas9技术,在武运粳7号背景下对TMS5基因进行编辑。【结果】 通过测序从22株T₀代中筛选获得6株纯合的突变体,分析发现6个纯合的突变体产生了相同的遗传变异,都在TMS5基因第2外显子44 bp和45 bp之间插入碱基“A”,导致翻译提前终止。通过细胞学观察和人工辅助授粉证实获得的株系花粉败育而雌配子正常发育。【结论】 这些结果表明对温敏不育基因TMS5基因进行编辑可以快速获得粳型温敏雄性不育系。

关键词: 粳稻, CRISPR/Cas9, 温敏雄性不育系

Abstract:

【Objective】The breeding of thermo-sensitive male sterile rice lines is a key step in the breeding of two-line hybrid rice. TMS5 is the key gene that modulates pollen fertility of rice at different temperatures and has been widely used in rice breeding. In order to accelerate the breeding process of thermo-sensitive sterility lines, 【Method】CRISPR/Cas9 technology was used to edit TMS5 with a japonica rice variety Wuyunjing 7 as a material. 【Results】Sequencing analysis showed that six homozygous mutants screened from 22 strains of T₀ generation produced the same genetic variation, and one base “A” was inserted between 44 bp and 45 bp of the second exon of TMS5 of all the six mutants, resulting in pre-maturation of translation. Cytological observation and artificial pollination confirmed that the pollen of tms5 mutant was aborted and the development of female gametes was normal. 【Conclusion】These results indicated that the thermo-sensitive male sterile lines could be obtained rapidly by knocking out the thermo-sensitive male sterile gene TMS5 in japonica rice.

Key words: japonica rice, CRISPR/Cas9, thermo-sensitive male sterile line

中图分类号:

杜茜, 费云燕, 王芳权, 许扬, 王军, 李文奇, 赵凌, 陈智慧, 梁国华, 周勇, 杨杰. 敲除TMS5基因获得温敏不育粳稻新材料[J]. 中国水稻科学, 2019, 33(5): 429-435.

Xi DU, Yunyan FEI, Fangquan WANG, Yang XU, Jun WANG, Wenqi LI, Ling ZHAO, Zhihui CHEN, Guohua LIANG, Yong ZHOU, Jie YANG. Thermo-sensitive Male Sterile Line Created by Editing TMS5 Gene in japonica Rice[J]. Chinese Journal OF Rice Science, 2019, 33(5): 429-435.

图/表 8

表1 本研究所用的引物

Table 1 Primers used in this study.

引物名称 Primer name	引物序列(5′-3′) Primer sequence(5'-3')
TMS5-U3-F	GGCAAGCTCAAGCCAGAGTATCT
TMS5-U3-R	AAACAGATACTCTGGCTTGAGCT
U-F	CTCCGTTTTACCTGTGGAATCG
U-R	CGGAGGAAAATTCCATCCAC
Uctcg-B1′	TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
gRcggt-BL	AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC
SP1	CCCGACATAGATGCAATAACTTC
SP2	GCGCGGTGTCATCTATGTTA
TMS5-TF	AGTTCCTCTTCATCTCCCAC
TMS5-TR	CAATCTCAGGCACCGTCAAT
Hpt-F	TCCGGAAGTGCTTGACATT
Hpt-R	GTCGTCCATCACAGTTTGC
Cas9-F	AGCGGCAAGACTATCCTCGACT
Cas9-R	TCAATCCTCTTCATGCGCTCCC

图1 TMS5靶序列位置和pYLCRISPR/Cas9-TMS5-1表达载体 A-靶序列在TMS5基因中的位置;红色方框代表PAM结构;B- pYLCRISPR/Cas9-TMS5-1表达载体。

Fig. 1. Schematic diagram of the targeted sequence in TMS5 and pYLCRISPR/Cas9-TMS5-1 vector. A, Target sequence in TMS5 gene; PAM structure is shown in red box; B, The expression vector of pYLCRISPR/Cas9-TMS5-1.

表2 T0代转基因植株中的突变率统计

Table 2 Mutation rate in T0 transgenic plants.

突变类型Mutation type	株数No. of plants	突变率Mutation rate / %
纯合突变Homozygous	6	27.3
杂合突变Heterozygous	3	13.6
双等位突变Bi-allelic	9	40.9
野生型Wild type	4	18.2

图2 野生型和突变体的氨基酸序列差异*为终止密码子。

Fig. 2. Amino acid sequence variance between wild type(WT) and tms5 mutant. * presents the termination codon.

图3 T1代突变系无T-DNA植株的PCR检测筛选 M-DNA标记;+：阳性对照;-：日本晴。

Fig. 3. Screening for T-DNA-free plants of T1 mutation lines by PCR analysis. M, DNA marker; +, Positive control; -, Nipponbare.

图4 野生型和突变体的表型

Fig. 4. Phenotype of wild type(WT) and the tms5 mutant.

图5 野生型和tms5突变体花粉育性比较 A-野生型花药形态;B-tms5突变体花药形态;C-野生型花粉染色;D-tms5突变体染色。

Fig. 5. Comparison of pollen fertility between wild type (WT) and tms5 mutant. A, Anthers of WT; B, Anthers of tms5 mutant; C, Pollen grains of WT stained with I2-KI; D, Pollen grains of tms5 stained with I2-KI.

图6 tms5与9311和武运粳7号杂交F1的结实情况 A-tms5突变体与9311杂交F1的结实情况;B-tms5突变体与武运粳7号杂交F1的结实情况。

Fig. 6. Seed setting of the F1 plants derived from tms5×9311 and tms5×Wuyunjing 7. A, Seed setting of the F1 plants derived from tms5×9311; B, The seed setting of the F1 plants derived from tms5×Wuyunjing 7.

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敲除TMS5基因获得温敏不育粳稻新材料

Thermo-sensitive Male Sterile Line Created by Editing TMS5 Gene in japonica Rice

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