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作物学报 ›› 2018, Vol. 44 ›› Issue (6): 844-851.doi: 10.3724/SP.J.1006.2018.00844

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于CRISPR/Cas9技术的水稻温敏不育基因tms5突变体的构建

黄忠明1,周延彪2,3,*(),唐晓丹3,赵新辉3,周在为3,符星学3,王凯4,史江伟3,李艳锋4,符辰建2,3,杨远柱1,2,3,4,*()   

  1. 1 湖南农业大学农学院, 湖南长沙410128
    2 袁隆平农业高科技股份有限公司 / 抗病虫水稻育种湖南省工程实验室, 湖南长沙410001
    3 湖南隆平高科种业科学研究院有限公司, 湖南长沙 410119
    4 湖南师范大学生命科学学院, 湖南长沙410081
  • 收稿日期:2017-10-25 接受日期:2018-03-26 出版日期:2018-06-12 网络出版日期:2018-04-08
  • 通讯作者: 周延彪,杨远柱
  • 基金资助:
    本研究由国家转基因生物新品种培育重大专项(2016ZX08001-004);湖南省重点研发计划项目资助(2016JC2026)

Construction of tms5 Mutants in Rice Based on CRISPR/Cas9 Technology

Zhong-Ming HUANG1,Yan-Biao ZHOU2,3,*(),Xiao-Dan TANG3,Xin-Hui ZHAO3,Zai-Wei ZHOU3,Xing-Xue FU3,Kai WANG4,Jiang-Wei SHI3,Yan-Feng LI4,Chen-Jian FU2,3,Yuan-Zhu YANG1,2,3,4,*()   

  1. 1 College of Agronomy, Hunan Agricultural University, Changsha 410128, Hunan, China
    2 Yuan Longping High-Tech Agriculture Co. Ltd., Hunan / Hunan Engineering Laboratory for Disease and Pest Resistance Rice Breeding, Changsha, 410001, Hunan, China
    3 Hunan Longping High-Tech Seeds Science Academy Co. Ltd., Changsha 410119, Hunan, China
    4 College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China
  • Received:2017-10-25 Accepted:2018-03-26 Published:2018-06-12 Published online:2018-04-08
  • Contact: Yan-Biao ZHOU,Yuan-Zhu YANG
  • Supported by:
    This study was supported by the Major Project of China on New Varieties of GMO Cultivation(2016ZX08001-004);the Key Research Program of Hunan Province(2016JC2026)

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摘要：

为创新优良温敏不育系, 促进两系杂交稻育种的发展, 我们以水稻温敏不育基因TMS5为编辑对象, 设计了由水稻U3启动子驱动、长20 bp的guide RNA (gRNA)靶点以靶向编辑 TMS5 基因的第1个外显子, 将靶点与表达载体pCAMBIA1301连接, 再用农杆菌介导法获得水稻转基因株系。提取T0代转基因株系的基因组DNA, 并对TMS5编辑位点附近的DNA片段进行PCR检测及测序分析。结果表明, T0代植株的突变率为63.89%, 其中纯合缺失突变率为34.78%。对T1代纯合缺失突变体的不育起点温度和农艺性状调查分析结果表明, 28℃是tms5突变体花粉育性的转换温度。大田试验结果表明, 与野生型相比, tms5突变体的结实率和单株重均显著降低。粳稻tms5突变体的获得为培育粳稻不育系奠定了材料基础。

关键词: 水稻, 基因编辑, CRISPR/Cas9, TMS5, 温敏不育

Abstract:

In order to create excellent temperature sensitive sterile line and promote the development of two line hybrid rice breeding. a 20 bp guide RNA (gRNA) was targeted to the first exon of TMS5, a thermo-sensitive genic male sterile gene as the editable object in present study, and driven by the rice U3 promoter. The target site of gRNA was ligated into the vector pCAMBIA1301, and its transgenic lines were obtained via Agrobacterium-mediated transformation. The genomic DNA of T0 transgenic lines was then extracted, and the TMS5 locus was detected by PCR and sequenced. The mutagenesis frequency for TMS5 was 63.89%, of which the frequency of homozygous deletion mutation was 34.78%. Furthermore, the fertility transition temperature and agronomic traits of T1 homozygous deletion mutants were investigated, showing that the fertility transition temperature of tms5 mutants was 28°C. Both seed setting rate and grain yields in tms5 mutants were significantly decreased compared with those in wild-type plants in paddy field condition. Together, the successful construction of tms5 mutants lays a material foundation for breeding the thermo-sensitive male sterile line of japonica rice.

Key words: rice, genome editing, CRISPR/Cas9, TMS5, thermo-sensitive genic male sterile

图1

TMS5靶点位置 红色的字母为靶点序列; 蓝色的字母为PAM序列。"

表1

本研究所用的引物"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
Target-1F TGGCGAACAGCGGCAAGTCATCGC
Target -1R AAACGCGATGACTTGCCGCTGTTC
M13F GTAAAACGACGGCCAG
GUS-JC-F CGTCCGTCCTGTAGAAACCC
GUS-JC-R GTGCGGATTCACCACTTGC
TMS5-CX-F TCCAACGCATAGCAGTAGTCG
TMS5-CX-R TGCCATCGTATCTCCGGTAAA

图2

CRISPR/Cas9-TMS5载体构建示意图 A: 18T-Cas9-TMS5-gRNA重组载体结构示意图; B: pCAMBIA1301载体的T-DNA片段; C: pCAMBIA1301-Cas9-TMS5-gRNA重组载体结构示意图。LB: 左边界; RB: 右边界。"

图3

酶切和PCR鉴定pCAMBIA1301-Cas9-TMS5-gRNA载体 A: Kpn I和Hind III酶切鉴定pCAMBIA1301-Cas9-TMS5-gRNA载体。箭头所示为CRISPR/Cas9系统元件的片段; B: 利用GUS-JC-F和GUS-JC-R引物PCR鉴定农杆菌中的pCAMBIA1301-Cas9-TMS5-gRNA载体。M: DL2000; 1: 18T-Cas9-TMS5-gRNA; 2: pCAMBIA1301; 3: pCAMBIA1301-Cas9-TMS5-gRNA。"

附图1

靶位点突变序列分析 红色的字母为靶点序列; 蓝色的字母为PAM序列; 绿色的字母为插入的碱基; +表示插入; WT表示野生型; Ho表示纯合突变; He表示杂合突变。"

图4

转基因植株的鉴定 A: PCR检测阳性转基因植株; B: GUS组织化学染色检测阳性转基因植株。M: DL2000; +: 阳性对照; 1-18: 转基因植株。"

图5

T0代转基因植株TMS5纯合突变类型和频率 A: 纯合突变类型序列比对; B: 纯合突变类型不同碱基插入的频率。红色字母为靶点序列; 蓝色字母为PAM序列; 绿色的字母为插入的碱基。"

图6

野生型和tms5突变体在不同温度处理条件下的花粉育性和结实率 数据表示平均值±方差。**表示tms5突变体与WT之间存在显著差异(P < 0.01)。"

表2

野生型(WT)和tms5突变体的农艺性状"

株系
Line		 株高
Plant height
(cm)		 千粒重
1000-grain weight
(g)		 有效穗
Panicle number
per plant		 结实率
Seed setting rate
(%)		 单株产量
Grain yield per plant
(g)
WT 78.96±3.56 20.82±0.97 9.3±1.87 72.21±3.57 17.30±1.09
tms5 78.57±3.87 21.20±0.88 9.85±1.79 61.60±3.43** 15.22±1.05**
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