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作物学报 ›› 2025, Vol. 51 ›› Issue (2): 334-346.doi: 10.3724/SP.J.1006.2025.32006

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

疣粒野生稻中OgXa13的克隆和功能研究

张正康1(), 苏延红1(), 阮孙美1, 张敏1, 张攀1, 张慧2, 曾千春2, 罗琼1,*()   

  1. 1云南农业大学云南生物资源保护与利用国家重点实验室, 云南昆明 650201
    2云南农业大学农学与生物技术学院, 云南昆明 650201
  • 收稿日期:2023-02-12 接受日期:2024-10-25 出版日期:2025-02-12 网络出版日期:2024-11-13
  • 通讯作者: 罗琼, E-mail: qiongbf@aliyun.com
  • 作者简介:张正康, E-mail: 1055909547@qq.com;
    苏延红, E-mail: 594355519@qq.com第一联系人：**同等贡献
  • 基金资助:
    NSFC-云南联合基金(U2102219);NSFC-云南联合基金(U1302261)

Cloning and functional study of OgXa13 in Oryza meyeriana

ZHANG Zheng-Kang1(), SU Yan-Hong1(), RUAN Sun-Mei1, ZHANG Min1, ZHANG Pan1, ZHANG Hui2, ZENG Qian-Chun2, LUO Qiong1,*()   

  1. 1Yunnan State Key Laboratory of Biological Resources Conservation and Utilization, Yunnan Agricultural University, Kunming 650201, Yunnan, China
    2College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan, China
  • Received:2023-02-12 Accepted:2024-10-25 Published:2025-02-12 Published online:2024-11-13
  • Contact: E-mail: qiongbf@aliyun.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    NSFC Yunnan United Fund(U2102219);NSFC Yunnan United Fund(U1302261)

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摘要：

白叶枯病是水稻生产中最严重的细菌性病害。优良抗病基因的挖掘和育种利用是防治该病害最经济有效的方法。疣粒野生稻(Oryza meyeriana)具有对白叶枯病高抗甚至免疫的特性, 是白叶枯病抗性基因资源的天然宝库。课题组在疣粒野生稻转录组和基因组测序的基础上, 从疣粒野生稻中克隆了水稻白叶枯抗性基因OsXa13的同源基因OgXa13的cDNA和含UTR区域的8908 bp基因组序列。序列分析结果显示, OgXa13基因由5个外显子和4个内含子组成, 与水稻中感病基因OsXa13的基因组结构和核心启动子序列均一致。OgXa13与水稻OsXa13的蛋白序列有21个氨基酸的差异, 其中4个氨基酸的替换差异位于MtN3.1结构域。过表达OgXa13的感病水稻TP309植株白叶枯病的抗性显著增强, 推测氨基酸序列差异导致了OgXa13与OsXa13蛋白功能的不同, OgXa13可作为一个显性白叶枯病抗性基因在育种中利用。利用CRISPR/Cas9敲除日本晴中感病基因OsXa13的T1代纯合株系对白叶枯病的抗性也明显增强, 表明通过CRISPR/Cas9编辑感病基因OsXa13是改良水稻对白叶枯病抗性的有效途径。该研究为水稻白叶枯病抗性育种提供了有价值的新基因资源和新信息。

关键词: 水稻, 疣粒野生稻, 白叶枯病抗性, 基因克隆, OgXa13

Abstract:

Bacterial blight is one of the most devastating bacterial diseases in rice production. The identification and utilization of resistance genes in rice breeding is the most economical and effective method for controlling this disease. Oryza meyeriana represents a valuable genetic resource due to its high resistance, and even immunity, to bacterial blight. In this study, we cloned the full-length cDNA and an 8908 bp genomic sequence of OgXa13, a homolog of OsXa13, from Oryza meyeriana using transcriptome and genome sequencing. Sequence analysis revealed that the gene structure of OgXa13 consists of five exons and four introns, mirroring the structure of rice OsXa13, and its core promoter sequence is identical to that of the rice susceptibility gene OsXa13. A total of 21 amino acid differences were observed between OgXa13 and OsXa13, with four key substitutions located in the MtN3.1 domain. Overexpression of OgXa13 and its introduction into TP309 plants via genetic transformation significantly enhanced resistance to bacterial blight. It is hypothesized that the amino acid sequence differences contribute to the distinct functions of the OgXa13 and OsXa13 proteins, suggesting that OgXa13 could be utilized as a dominant resistance gene in rice breeding. Furthermore, knockout of the OsXa13 gene using CRISPR/Cas9 in Nipponbare T1 homozygous lines also significantly enhanced resistance to bacterial blight, demonstrating that editing the susceptibility gene OsXa13 through CRISPR/Cas9 is an effective strategy for improving resistance. This study provides a valuable genetic resource and new insights for breeding rice with enhanced resistance to bacterial blight.

Key words: rice, Oryza meyeriana, bacterial blight resistance, gene cloning, OgXa13

图1

4个gRNA靶点在OsXa13基因上的位置 PAM: CRISPR/Cas对特定短DNA序列的识别, 标红处为PAM序列。"

表1

寡核苷酸序列"

Oligo名称
Oligo name		 正向序列
Forward sequence (5′-3′)		 反向互补序列
Reverse complementary sequence (5′-3′)
Oligo-Target1 TGTGTGTCACCCTCTCCGGTGTTGC AAACGCAACACCGGAGAGGGTGACA
Oligo-Target2 TGTGTGGGGTACAGCTCGGTGCCGT AAACACGGCACCGAGCTGTACCCCA
Oligo-Target3 TGTGTGCTACCTCGTCTACGCGCCG AAACCGGCGCGTAGACGAGGTAGCA
Oligo-Target4 TGTGTGAGCCGAGGAACACGGCCGT AAACACGGCCGTGTTCCTCGGCTCA

表2

转基因植株鉴定引物"

引物Primers 引物序列Primer sequences (5′-3′) 用途Purpose
HYG F: GCTTCTGCGGGCGATTTGTGT
R: GGTCGCGGAGGCTATGGATGC		 潮霉素检测引物
Hygromycin detection primer
OgXa13-ox F: TAGTGGAAAAGGAAGGTGGCTCC
R: TGGGGACGAAGTAGAGCGTGAC		 过表达阳性株系鉴定
Identification of positive overexpression lines
OgXa13 F: GGCAAGCTGCTCTAGCCAATAC
R: CAAGGGGAACAGCTAACAAGTGC		 OgXa13基因阳性株系鉴定
Identification of transgenic OgXa13 gene-positive lines
Target1 F: GCTAGAGAGGAAGGCTTAAG
R: CAATGCAATGGTGCAGACAAC		 靶点1测序引物
Target 1 sequencing primer
Target2/3 F: CATCATCTCCTTCCTGGTGTTCC
R: GACGGAGAGCCCGATCGGCATG		 靶点2和靶点3测序引物
Target 2 and target 3 sequencing primers
Target4 F: CATGCCGATCGGGCTCTCCGTC
R: CACCACCTCGACCTTGTGCAC		 靶点4测序引物
Target 4 sequencing primer

图2

OgXa13与OsXa13的基因结构示意图 A: OgXa13的基因结构示意图; B: OsXa13的基因结构示意图。外显子用黑色方框表示, 内含子用白色方框表示。"

图3

OgXa13和OsXa13蛋白结构分析 A: OgXa13与OsXa13的蛋白结构示意图, 灰色方框代表MtN3_slv结构域; 黑色细线标注的是OgXa13和OsXa13在MtN3_slv结构域上的差异氨基酸及其位点; B: OgXa13蛋白的跨膜预测图; C: OsXa13蛋白的跨膜预测图。"

图4

过表达OgXa13的TP309植株白叶枯病抗性 A: OgXa13过表达植株接种白叶枯病菌PXO99后14 d叶片发病症状; B: 过表达TP309植株OgXa13基因鉴定; C: 转OgXa13基因T1代纯合植株接种白叶枯病菌PXO99后14 d病斑长度; D: 转OgXa13基因T1代纯合植株接种白叶枯病菌PXO99后14 d病斑长度占叶片总长度百分比; E: 转OgXa13基因T1代纯合植株叶片中OgXa13的表达水平。*, P > 0.05; **, P < 0.01。"

图5

转OgXa13基因植株的白叶枯病抗性 A: 转基因植株接种白叶枯病菌PXO99后14 d叶片发病症状; B: 转基因植株的基因特异性引物鉴定; C: 阳性转基因T1代纯合植株接种PXO99 0 h和48 h后叶片中OgXa13的表达水平; D: 转基因植株接种白叶枯病菌PXO99后14 d病斑长度占叶片总长度百分比; E: 转基因T1代纯合植株接种白叶枯病菌PXO99后14 d病斑长度。*, P > 0.05; **, P < 0.01。"

图6

转OgXa13基因T2代水稻植株的白叶枯病抗性 A: T2转基因植株接种白叶枯病菌PXO99后21 d叶片发病症状; B: 转基因植株的基因特异性引物鉴定; C: T2代转基因植株接种白叶枯病菌PXO99后21 d后病斑长度; D: 转基因植株接种白叶枯病菌PXO99后21 d病斑长度占叶片总长度百分比; E: T2代转基因纯合植株接种PXO99后21 d菌物量统计。*, P > 0.05; **, P < 0.01。"

图7

OsXa13的CRISPR/Cas9敲除株系的白叶枯病抗性 A: 日本晴与T1代敲除株系突变位点核酸序列及氨基酸序列比对; B: 日本晴和3个纯合敲除株系接种白叶枯病菌PXO99后14 d病斑长度; C: 野生型日本晴和3个纯合敲除株系接种白叶枯病菌PXO99后14 d病斑长度; D: 野生型日本晴和3个纯合敲除株系接种白叶枯病菌PXO99后14 d病斑长度占叶片总长度百分比。Nip: 日本晴; Xa13-KO: Xa13敲除株系。*, P > 0.05; **, P < 0.01。"

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doi: 10.1093/mp/sst035 pmid: 23430047
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