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作物学报 ›› 2023, Vol. 49 ›› Issue (2): 392-401.doi: 10.3724/SP.J.1006.2023.21003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

栽培小麦Brock中抗白粉病相关基因TaRPP13-1B的克隆及功能分析

刘晓颖1(), 张驰1, 王雪晴1, 杨晨晓1, 王光钰1, 卞云迪1, 方芳2, 王颖2, 王振英1,*()   

  1. 1天津师范大学生命科学学院 / 天津市动植物抗性重点实验室, 天津 300387
    2天津市宝坻区林业发展服务中心, 天津 301899
  • 收稿日期:2022-01-16 接受日期:2022-05-05 出版日期:2022-05-25 网络出版日期:2022-05-25
  • 通讯作者: 王振英
  • 作者简介:E-mail: skylxy@tjnu.edu.cn
  • 基金资助:
    国家自然科学基金项目(31071671);天津市科技支撑项目(18YFZCNC01100);天津市科技支撑项目(17JCZDJC34100)

Cloning and functional analysis of TaRPP13-1B gene related to powdery mildew resistance in wheat cultivar Brock

LIU Xiao-Ying1(), ZHANG Chi1, WANG Xue-Qing1, YANG Chen-Xiao1, WANG Guang-Yu1, BIAN Yun-Di1, FANG Fang2, WANG Ying2, WANG Zhen-Ying1,*()   

  1. 1College of Life Sciences, Tianjin Normal University, Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin 300387, China
    2Forestry Development Service Center of Baodi District, Tianjin 301899, China
  • Received:2022-01-16 Accepted:2022-05-05 Published:2022-05-25 Published online:2022-05-25
  • Contact: WANG Zhen-Ying
  • Supported by:
    National Natural Science Foundation of China(31071671);Tianjin Science and Technology Program(18YFZCNC01100);Tianjin Science and Technology Program(17JCZDJC34100)

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摘要：

白粉病是小麦生产中的主要病害之一, 发掘抗病基因并实现抗病基因转育是提高作物抗病性的最经济有效的方法。本研究克隆了一个位于小麦染色体1B上、具有典型CC、NBS和LRR结构域的基因TaRPP13-1B。接种白粉菌后, TaRPP13-1B基因在抗病小麦Brock和BJ-1中表达量虽然出现上下调波动, 但平均表达水平一直高于感病小麦品种京411。采用病毒诱导的基因沉默和转基因过表达技术进行功能分析, 发现抑制目标基因表达, 抗病小麦品种Brock对白粉菌的抗性显著降低; 过表达TaRPP13-1B的转基因小麦津强5号对白粉菌抗性明显提高。说明TaRPP13-1B基因参与小麦抗白粉病的防御反应过程。该研究为小麦抗病品种的选育提供了有价值的备选基因。

关键词: 小麦, NBS-LRR类基因, TaRPP13-1B, 抗白粉病

Abstract:

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a severe wheat disease in China. Cloning and pyramiding of different resistance genes to improve crop disease resistance is one of the most cost-effective methods. In this study, TaRPP13-1B on chromosome 1B, which encodes the CC, NB-ARC, and LRR domains, was isolated from common wheat. The relative expression level of TaRPP13-1B in Brock and BJ-1 fluctuated after Bgt inoculation, but the average expression levels were always higher than the susceptible wheat Jing 411. The function of TaRPP13-1B was elucidated by virus-induced gene silencing (VIGS) and overexpression transgenic technique. Silencing of TaRPP13-1B resulted in decreased disease resistance in Brock. Overexpression of TaRPP13-1B improved disease resistance in the transgenic wheat seedlings of Jinqiang 5 cultivar. The above results demonstrated that TaRPP13-1B was involved in the defense response of wheat to powdery mildew, which provided valuable genetic resources for breeding of resistant varieties.

Key words: wheat, NBS-LRR gene, TaRPP13-1B, powdery mildew resistance

表1

本研究的引物及序列"

引物Primer name 序列Sequence (5'-3') 用途Gene usage
TaRPP13-1B-F/R F: ATGGAGTTAGCCGTGGGCGC
R: TCAATCATGATACTCGTTAATAATTAGGG		 cDNA克隆 Isolation of cDNA
RPP13-1B-GSP1 F: GCCTGGGCCAACTACGTTTCAAGTC 3'RACE第1轮PCR 1st run PCR of 3'RACE
RPP13-1B-GSP2 F: AAGCCTTCGAGGGACAGACG 3'RACE 第2轮PCR 2nd run PCR of 3'RACE
RPP13-1B-5RACE-1 R: AATCTCTGTCTCCCTTATGTCTAGGGTC 5'RACE第1轮PCR 1st run PCR of 5'RACE
RPP13-1B-5RACE-2 R: ATGACTTGAAACGTAGTTGGCCCAGG 5'RACE第2轮PCR 2nd run PCR of 5'RACE
TaRPP13-1B-ORF F: ATGGAGTTAGCCGTGGGCGC 全长cDNA克隆 Isolation of full-length cDNA
R: TCAATCATGATACTCGTTAATAATTAGGG
Actin-F/R F: TACTCCCTCACAACAACCG qRT-PCR
R: AGAACCTCCACTGAGAACAA
qRPP13-1B F: CCTACGACATCGAGGACTGC qRT-PCR
R: CGTACAGAGTCCTGCGGATC
RPP13-1B-V F: CTAGCTAGCAAAATGGATTACGGAAGGG BSMVγ: TaRPP13-1B载体构建
Construction of BSMVγ: TaRPP13-1B vector
R: CTAGCTAGCCGACCAGTAGCCACCAAC
RPP13-1B-BglF/BstE R F: GAAGATCTATGGAGTTAGCCGTGGGCGC TaRPP13-1B过表达载体构建
Construction of TaRPP13-1B overexpression vector
R: GGTAACCTCAATCATGATACTCGTTAATAATTAGGG
RPP13-1B-R(35S) TCTTCTGAAAAGTGCTGCCTG 鉴定转基因小麦
Identification of transgenic wheats

图1

TaRPP13-1B基因的结构域分析 A: TaRPP13-1B的基因结构; B: TaRPP13-1B蛋白的结构域; C: 不同植物中RPP13蛋白的进化树。"

图2

TaRPP13-1B和TaRPP13-3的同源性分析"

图3

TaRPP13-1B基因的白粉菌诱导表达分析 Jing 411: 感病小麦品种京411; BJ-1: 京411抗病近等基因系; Brock: 抗病小麦品种; Student’s t测验, *: P < 0.05; **: P < 0.01。"

图4

沉默TaRPP13-1B后小麦抗病性变化 A: Brock接种BSMV病毒后表型; B: 沉默叶片接种白粉菌E09的表型观察; C: 各实验组中TaRPP13-1B基因的相对表达量(*: P < 0.05)。"

图5

TaRPP13-1B基因沉默Brock叶片上白粉菌孢子的显微观察 LA: 分瓣型畸形附着胞; SA: 纤细型附着胞; AGT: 喙型附着胞; SH: 次生菌丝; BC: 念珠状分生孢子。"

图6

TaRPP13-1B T0代转基因小麦的PCR检测 M: DL2000 marker; 1: 1301对照组; 2: 质粒阳性对照; 3~14: T0代转基因植株。"

图7

TaRPP13-1B在T1代转基因小麦中的表达量分析 C: 对照组; 1~11: T1-1~T1-11 转基因单株(**: P < 0.01)。"

图8

过表达TaRPP13-1B基因提高小麦对白粉菌E09的抗性 A: 转基因小麦在白粉菌E09侵染7 d后的叶片表型; B: 白粉菌E09侵染下的小麦叶片病斑面积(**: P < 0.01)。"

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doi: 10.1105/tpc.113.109942
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