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作物学报 ›› 2020, Vol. 46 ›› Issue (8): 1157-1165.doi: 10.3724/SP.J.1006.2020.92059

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用CRISPR/Cas9技术编辑水稻温敏不育基因TMS5

陈日荣1,周延彪2,3,王黛君5,赵新辉3,唐晓丹3,许世冲4,唐倩莹3,符星学3,王凯3,刘选明1,*(),杨远柱1,2,3,4,*()   

  1. 1湖南大学生物学院/植物功能基因组学与发育调控湖南省重点实验室, 湖南长沙410082
    2袁隆平农业高科技股份有限公司/农业农村部南方水稻品种创制重点实验室, 湖南长沙410001
    3湖南亚华种业科学研究院, 湖南长沙 410119
    4华中农业大学植物科学技术学院, 湖北武汉 430070
    5湖南师范大学生命科学学院, 湖南长沙 410081
  • 收稿日期:2019-11-09 接受日期:2020-04-15 出版日期:2020-08-12 网络出版日期:2020-04-26
  • 通讯作者: 刘选明,杨远柱
  • 作者简介:E-mail: 641605686@qq.com。
  • 基金资助:
    国家自然科学基金项目(31901516);国家转基因生物新品种培育重大专项(2016ZX08001-004);湖南省自然科学青年基金项目(2019JJ50414)

CRISPR/Cas9-mediated editing of the thermo-sensitive genic male-sterile gene TMS5 in rice

CHEN Ri-Rong1,ZHOU Yan-Biao2,3,WANG Dai-Jun5,ZHAO Xin-Hui3,TANG Xiao-Dan3,XU Shi-Chong4,TANG Qian-Ying3,FU Xing-Xue3,WANG Kai3,LIU Xuan-Ming1,*(),YANG Yuan-Zhu1,2,3,4,*()   

  1. 1Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation/College of Biology, Hunan University, Changsha 410082, Hunan, China
    2Yuan Longping High-Tech Agriculture Co. Ltd./Key Laboratory of Rice Germplasm Enhancement in Southern China, Ministry of Agriculture and Rural Affairs, Changsha 410001, Hunan, China
    3Yahua Seeds Science Academy of Hunan, Changsha 410119, Hunan, China
    4College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
    5College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China
  • Received:2019-11-09 Accepted:2020-04-15 Published:2020-08-12 Published online:2020-04-26
  • Contact: Xuan-Ming LIU,Yuan-Zhu YANG
  • Supported by:
    National Natural Science Foundation of China(31901516);National Major Project for Developing New GM Crops(2016ZX08001-004);Project of Hunan Natural Science Youth Foundation(2019JJ50414)

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摘要：

tms5是生产上应用最广泛的温敏不育基因。为创制新型水稻温敏核不育系, 利用CRISPR/Cas9基因编辑技术将6个优异的粳稻和4个优异的籼稻的TMS5基因敲除, 获得了温敏核不育系。比较发现, 粳稻温敏不育系ZG75S、CYGS、YG0618S、ZG07S、T0361S、7679S的不育起点温度在28~32°C之间, 籼稻温敏核不育系2537S、6150S、6379S的不育起点温度在24~28°C之间, 而籼稻温敏核不育系1109S的不育起点温度低于23.5°C。说明不同遗传背景材料获得的tms5突变体的不育起点温度不一样, 通过TMS5的敲除可能获得不育起点温度较低的温敏核不育系。利用1109S与优质父本8048选配出优质高产杂交稻组合1109S/8048。大田试验表明, 1109S/8048比区试对照丰两优4号增产13.1%。温敏核不育系1109S及高产杂交稻组合1109S/8048的创制为高产育种提供了新的途径。

关键词: 水稻, CRISPR/Cas9, 温敏不育, TMS5, 杂交组合

Abstract:

Thermo-sensitive genic male-sterile (TGMS) gene tms5 is most widely used in the two-line hybrid breeding system in China. To develop novel rice thermo-sensitive male sterile lines, we knocked out the TMS5 genes of six elite japonica and four indica rice varieties by using CRISPR/Cas9 gene editing technology. By analyzing the critical sterility-inducing temperature (CIST) of the newly TGMS lines, it was found that the CIST of japonica TGMS lines ZG75S, CYGS, YG0618S, ZG07S, T0361S, and 7679S were between 28°C and 32°C, the CIST of indica TGMS lines 2537S, 6150S and 6379S were between 24°C and 28°C, and the CIST of indica TGMS line 1109S was lower than 23.5°C. These results indicated that the CIST of tms5 mutant from different genetic background materials was different. The TGMS lines with lower CIST could be obtained by knocking out the TMS5 from different genetic background materials. A hybrid rice combination 1109S/8048 had high quality and high yield. The yield of 1109S/8048 was 13.1% higher than that of Fengliangyou 4. The creation of the TGMS 1109S and the high-yield cross combination 1109S/8048 provides a new way for high-yield breeding.

Key words: rice, CRISPR/Cas9, thermo-sensitive genic male sterile, TMS5, cross combination

图1

CRISPR/Cas9-TMS5载体构建示意图 A: TMS5靶点位置。红色字母为靶点序列, 蓝色字母为PAM序列。B: pCAMBIA1301-Cas9-TMS5-gRNA重组载体结构示意图。LB: 左边界; RB: 右边界。"

表1

本研究所用的引物"

引物名称 Primer name 引物序列 Primer sequence (5′-3′)
TMS5-target-F TGGCCAGCGGCAAGTCATCGCCGG
TMS5-target-R AAACCCGGCGATGACTTGCCGCTG
M13F GTAAAACGACGGCCAG
GUS-F CGTCCGTCCTGTAGAAACCC
GUS-R GTGCGGATTCACCACTTGC
TMS5-CX-F TCCAACGCATAGCAGTAGTCG
TMS5-CX-R TGCCATCGTATCTCCGGTAAA
Cas9-F ACCGAGGGAATGAGAAAGCC
Cas9-R CCTTCTGGGTGGTCTGGTTC
HPT-F GAAGTGCTTGACATTGGGAGT
HPT-R AGATGTTGGCGACCTCGTATT

图2

T0代转基因植株的鉴定 ZG75(A)、CYG(B)、YG0618(C)、ZG07(D)、T0361(E)、7679(F)、2537(G)、6150(H)、6379(I)、1109(J)阳性转基因植株的PCR鉴定(上)和GUS组织化学染色(下)。M: DNA marker; 1~10: 转基因植株。"

图3

T0代纯合突变类型 红色字母为靶点序列; 蓝色字母为PAM序列; 绿色字母为插入的碱基; 横线表示碱基缺失; +表示插入; -表示缺失; WT表示野生型。"

附图1

PCR鉴定T1代无选择标记基因的突变株 ZG75(A)、CYG(B)、YG0618(C)、ZG07(D)、T0361(E)、7679(F)、2537(G)、6150(H)、6379(I)、1109(J)非转基因植株的PCR鉴定。M: DNA marker; WT: 野生型; 1: 阳性对照; 2~6: GUS染色不呈蓝色的T1代植株。"

图4

tms5突变体在不同温度处理条件下的花粉育性 A: tms5粳稻突变体的花粉育性; B: tms5籼稻突变体的花粉育性; C: 1109S在冷灌处理条件下的花粉育性。"

图5

1109S/8048杂交组合的表型鉴定 A: 1109S/8048杂交组合的表现, Bar = 20 cm; B: 1109S/8048杂交组合穗的表现, Bar = 5 cm。"

表2

杂交组合的农艺性状"

品种
Variety		 株高
Plant height (cm)		 有效穗
Panicle number per plant		 千粒重
1000-grain weight (g)		 穗长
Length of main panicle (cm)		 穗粒数
Grain number per panicle		 结实率
Seed-setting rate (%)		 产量
Grain yield
(kg hm-2)
8048 110.4±3.5 10.7±1.0 16.8±1.6 27.5±1.0 229.3±15.8 74.6±8.2 8857.5±526.5
丰两优4号
Fengliangyou 4		 133.1±4.5 11.5±1.2 27.5±2.1 25.3±1.1 193.5±14.6 75.7±5.6 9835.5±387.0
1109S/8048 120.6±4.0** 13.5±2.0** 22.6±1.5** 30.7±1.5** 258.4±23.6** 75.9±4.5 11124.0±279.0**
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