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作物学报 ›› 2020, Vol. 46 ›› Issue (9): 1332-1339.doi: 10.3724/SP.J.1006.2020.02013

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻白叶枯病感病相关基因Xig1的分子鉴定及抗病资源创制

郑凯丽1(), 纪志远1(), 郝巍1, 唐永超1, 韦叶娜1,2, 胡运高2, 赵开军1, 王春连1,*()   

  1. 1 中国农业科学院作物科学研究所 / 农作物基因资源与基因改良国家重大科学工程, 北京 100081
    2 西南科技大学水稻研究所, 四川绵阳621010
  • 收稿日期:2020-02-27 接受日期:2020-04-15 出版日期:2020-09-12 网络出版日期:2020-05-07
  • 通讯作者: 王春连
  • 作者简介:郑凯丽, E-mail: zhkl111007@163.com|纪志远, E-mail: jizhiyuan@caas.cn
  • 基金资助:
    本研究由国家自然科学基金项目(31571640);中国科协青年人才托举工程资助(2017QNRC001)

Molecular identification of rice bacterial blight susceptible gene Xig1 and creation of disease resistant resources

ZHENG Kai-Li1(), JI Zhi-Yuan1(), HAO Wei1, TANG Yong-Chao1, WEI Ye-Na1,2, HU Yun-Gao2, ZHAO Kai-Jun1, WANG Chun-Lian1,*()   

  1. 1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing 100081, China
    2 Rice Research Institute, Southwest University of Science and Technology, Mianyang 621010, Sichuan, China
  • Received:2020-02-27 Accepted:2020-04-15 Published:2020-09-12 Published online:2020-05-07
  • Contact: Chun-Lian WANG
  • Supported by:
    National Natural Science Foundation of China(31571640);Young Elite Scientist Sponsorship of China Association for Science and Technology(2017QNRC001)

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摘要：

水稻白叶枯病相关基因Xig1在感病亲本IR24中受白叶枯病菌的诱导表达。本研究通过克隆与比较Xig1的序列后发现, 抗病野生稻导入系W6023与感病亲本IR24中Xig1等位基因的差异主要集中在启动子区。水稻原生质体观察XIG1在细胞中的定位, 显示XIG1定位于细胞质中。利用CRISPR/Cas9系统对感病籼稻品种IR24的Xig1靶点进行基因组编辑, 获得并评价了多个Xig1定点突变株系对白叶枯菌的抗性。与野生型相比, 8个IR24的Xig1基因定点突变株系对水稻白叶枯病的抗性得到明显提高, 而农艺性状无显著差异。Xig1是新发现的水稻白叶枯病感病基因, 该基因在水稻白叶枯病感病性中贡献的研究, 不仅为创制新的抗病资源提供理论指导, 还将丰富和加深我们对植物先天免疫系统的认知。

关键词: 水稻, 白叶枯病, Xig1, CRISPR/Cas9

Abstract:

The rice bacterial blight susceptible gene Xig1 (Xoo-induced-gene 1), was found highly induced by Xanthomonas oryzae pv. oryzae (Xoo) in susceptible parent line IR24 but not in introgression line W6023. In this study, we cloned and sequenced Xig1 alleles isolated from W6023 and IR24, and the main difference in nucleotide sequences between Xig1 alleles of W6023 and IR24 was in the promoter region. Subcellular localization in rice protoplast indicated that XIG1 localized to cytoplasm. CRISPR/Cas9-based genome editing technologies were also applied to knock out Xig1 in IR24. Eight Xig1-edited mutant lines showed a higher resistance to Xoo, and all agronomic traits surveyed are consistent. Xig1 is a novel susceptible gene for bacterial blight in rice. The studying of the mechanism of Xig1 to Xoo in rice will not only provide a theorical guidance for disease resistance resource, but also benefit the understanding of plant innate immunity.

Key words: rice, bacterial blight, Xig1, CRISPR/Cas9

图1

接种P6后IR24和W6023中Xig1基因相对表达量"

表1

Xig1IR24基因启动子区内顺式元件特征分析"

顺式作用元件
Cis-acting element		 核心序列
Core sequence		 功能
Function		 数量
Amount
GARE-motif AAACAGA 赤霉素反应元件Gibberellin response-related elements 1
GT1-motif GGTTAA 光响应元件Light response elements 2
MBS CGGTCA, CAACTG MYB结合位点、参与干旱诱导
MYB binding sites involved in drought-inducibility		 4
Skn-1motif GTCAT 胚乳表达所需元件Elements related to endosperm expression 4
TA-rich region TATATATATATATATATATATA 增强子Enhancer 1
TATA-box TATA, TTA, TAATA 核心启动子元件Core promoter element 21
TC-rich repeats GTTTTCTTAC, ATTTTCTCCA 防御和应激反应元件
Elements involved in defense and stress responsiveness		 2
TGA-element AACGAC 生长素应答元件Auxin response elements 1

图2

YFP标记的XIG1::YFP融合蛋白的亚细胞定位结果 YFP: 黄色荧光; Bright field: 可见光; Merge: 黄色荧光与可见光叠加。"

图3

T0代转基因材料的分子检测 M: 分子量标记; WT: IR24 (阴性对照); 1~32: T0代转基因植株。"

图4

17个T2代基因编辑纯合株系靶点序列 大写加粗字母为PAM核苷酸序列; 下画线序列为靶点序列; 红色破折号表示核苷酸缺失; 红色小写字母表示插入的核苷酸; 斜体大写字母为起始密码子。序列右侧的数字代表各靶位点突变类型及突变所涉及的核苷酸数。“-”和“+”分别表示缺失和插入所指示的核苷酸数。100 bp为此序列中未显示的一段碱基序列。"

图5

8个T2转基因抗病株系抗病反应 A, C: 分别为接种7 d和14 d时8个转基因株系与对照IR24病斑长度症状图。B, D: 分别为接种7 d和14 d时8个转基因株系与对照IR24病斑长度柱形图。**代表极显著差异(P < 0.01)。箭头: 接种的IR24叶片。"

表2

8个基因编辑株系的农艺性状分析"

基因编辑株系
Lines		 株高
Plant height
(cm)		 分蘖数
Number of tillers		 穗粒数
Number of grains
per panicle		 结实率
Seed-setting rate
(%)		 千粒重
Thousand-seed weight (g)		 籽粒产量
Grain yield (g)
IR24 87.2±2.1 a 10.8±1.3 a 112.7±12.6 a 82.9±1.9 a 24.8±0.8 a 24.5±2.9 a
Xig1-3 87.1±1.6 a 11.9±1.7 a 110.9±9.9 a 80.2±1.4 a 24.4±1.0 a 26.0±4.5 a
Xig1-6 87.8±2.6 a 11.8±1.9 a 108.8±5.5 a 80.0±1.7 a 25.9±0.9 a 26.7±2.6 a
Xig1-12 86.7±2.5 a 10.6±0.9 a 114.5±10.9 a 81.2±1.5 a 24.6±0.7 a 24.9±6.0 a
Xig1-15 86.5±1.5 a 10.5±1.7 a 114.6±11.3 a 83.6±0.4 a 24.9±1.8 a 25.2±2.5 a
Xig1-20 86.7±1.5 a 11.3±1.6 a 116.7±8.9 a 84.1±1.1 a 25.1±1.2 a 28.0±3.7 a
Xig1-21 87.6±2.3 a 10.4±1.5 a 123.0±10.0 a 82.7±2.2 a 25.9±1.3 a 27.2±2.0 a
Xig1-24 88.2±1.9 a 13.2±3.2 a 97.4±4.3 a 83.2±3.3 a 25.2±0.5 a 26.8±2.1 a
Xig1-26 88.4±1.7 a 10.2±2.2 a 118.0±12.9 a 84.2±3.3 a 25.5±0.8 a 25.6±3.1 a
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