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作物学报 ›› 2019, Vol. 45 ›› Issue (8): 1158-1165.doi: 10.3724/SP.J.1006.2019.81017

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦类受体蛋白激酶基因TaPK3A的克隆与抗纹枯病功能初步分析

苏强,荣玮,张增艳()   

  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/农业部麦类生物学与遗传育种重点实验室, 北京 100081
  • 收稿日期:2018-09-30 接受日期:2019-01-19 出版日期:2019-08-12 网络出版日期:2019-07-16
  • 通讯作者: 张增艳
  • 作者简介:E-mail: 243939427@qq.com
  • 基金资助:
    本研究由国家自然科学基金项目资助(31771789)

Cloning and functional analysis of wheat receptor-like kinase gene TaPK3A

SU Qiang,RONG Wei,ZHANG Zeng-Yan()   

  1. National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Crop Genetic and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2018-09-30 Accepted:2019-01-19 Published:2019-08-12 Published online:2019-07-16
  • Contact: Zeng-Yan ZHANG
  • Supported by:
    This study was support by the National Natural Science Foundation of China(31771789)

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摘要：

小麦纹枯病已成为我国小麦生产的重要限制因素。研究小麦防御反应分子基础, 发掘有效的抗病基因是小麦抗纹枯病育种突破的前提。本研究从抗纹枯病小麦品系CI12633中克隆出一个类受体蛋白激酶(receptor-like protein kinase, RLK)基因TaPK3A, 并对其表达特性及抗纹枯病功能进行了分析和验证。TaPK3A基因的开放阅读框长度为1983 bp, 编码660个氨基酸组成的类受体蛋白激酶。TaPK3A在抗纹枯病小麦品系CI12633中的表达受禾谷丝核菌的诱导而显著上调; TaPK3A在根、茎、叶、穗中都有表达, 以叶中的表达量最高; TaPK3A的表达受植物激素水杨酸诱导最为显著。利用大麦条形花叶病毒(barley stripe mosaic virus, BSMV)诱导的基因沉默(virus-induced gene silencing, VIGS)技术, 降低抗纹枯病小麦CI12633中TaPK3A的转录水平, 再接种禾谷丝核菌WK207进行纹枯病抗性鉴定。结果显示, 与对照植株相比, TaPK3A转录量下降的CI12633植株对纹枯病的抗性显著降低, 说明TaPK3A是小麦防御纹枯病反应所需的。

关键词: 类受体蛋白激酶TaPK3A, 小麦纹枯病, 抗病反应, 病毒诱导的基因沉默

Abstract:

Wheat sharp eyespot has become an important limiting factor of wheat production in China. The precondition for wheat sharp eyespot resistant breeding is to study the molecular basis of wheat defense response and to identify effective resistance genes. In this study, a wheat receptor-like kinase (RLK) gene, named TaPK3A, was cloned from sharp eyespot-resistant wheat line CI12633, and the expression and function of the TaPK3A gene were analyzed. TaPK3A contains an open reading frame with 1983 bp length. It encodes a protein kinase that is consisted of 660 amino acids. RT-qPCR analysis showed that the expression of TaPK3A in sharp eyespot-resistant wheat line CI12633 was significantly induced by the pathogen of sharp eyespot (Rhizoctonia cerealis). The TaPK3A gene was expressed in all the tissues, with the highest expression level in the leaves. The expression of TaPK3A was significantly up-regulated by salicylic acid. By means of barley stripe mosaic virus (BSMV) based virus-induced gene-silencing (VIGS), TaPK3A was silenced in CI12633 plants. After R. cerealis (WK207) inoculation, TaPK3A-silenced CI12633 plants displayed a significant decrease in resistance to R. cerealis infection compared with BSMV: GFP-infected CI12633 plants (control). These results suggested that TaPK3A is required for wheat defense response to sharp eyespot.

Key words: receptor-like kinase TaPK3A, wheat sharp eyespot, resistance response, virus-induced gene silencing

图1

RIL中TaPK3A的表达分析 A: 抗感材料RNA-Seq 数据中TaPK3A的表达量; B: RT-qPCR验证TaPK3A的表达量; R代表RIL中抗纹枯病小麦株系, S代表RIL中感纹枯病株系; R0/S0表示在0 h时R中和S中TaPK3A的表达量的比值; R4/S4表示在4 d时R中和S中TaPK3A的表达量的比值; R10/S10表示在10 d时R中和S中TaPK3A的表达量的比值。"

图2

TaPK3A蛋白的系统进化树分析 用MEGA7对TaPK3A进化树分析, 五角星标注的是TaPK3A蛋白, 竖线内为同一亚家族蛋白。AtCERK1 (NP_566689.2)、AtLecRK-VI.2 (Q9M021.1)、AtLPK1 (NP_567233.1)、AtPSKR1 (OAP07882.1)、AtWAK1 (OAP13193.1)、AtWAKL10 (NP_178086.1)来自拟南芥; OnN25-1 (AHW98543.1)来自野生稻; NbLRK1 (BAG68210.1)来自本氏烟; OsWAK1 (BAG89532.1)、OsCERK1 (A0A0P0XII1.1)、OsXa26 (AAR08150.1)来自水稻; Sbds1 (BAM45642.1)来自高粱; SiBti9 (ADL16642.1)来自谷子; TaLRK10 (AAC49629.1)来自小麦; ZmPK3 (CAA09029.1)来自玉米。"

图3

小麦品系CI12633中TaPK3A基因的表达分析 A: TaPK3A基因在抗纹枯病小麦CI12633中的表达分析, 横坐标表示禾谷丝核菌侵染小麦CI12633后0 h、2 d、4 d、7 d、10 d、14 d和21 d时的样品。B: CI12633小麦不同组织中TaPK3A受禾谷丝核菌的诱导表达分析。C: TaPK3A基因响应 SA、JA、ABA和 ET的表达分析, 横坐标表示不同植物激素处理小麦CI12633后0 h、0.5 h、1 h、6 h和24 h时的样品。t检验分析TaPK3A表达量的差异显著性, *P < 0.05; **P < 0.01。"

图4

BSMV CP、TaPK3A基因的检测及小麦花叶和纹枯病表型 A: 半定量PCR检测CP的转录表达, 扩增26个循环; B: 对接种BSMV: GFP和BSMV: TaPK3A 10 d后的植株RT-qPCR检测TaPK3A的表达, **P < 0.01 (t检验); C: 接种BSMV后10 d, 小麦叶片呈现出BSMV病毒斑; D: 接种禾谷丝核菌后30 d小麦茎秆上纹枯病斑, IT: 病级"

表1

TaPK3A沉默及对照小麦的纹枯病鉴定"

基因型
Genotype		 病级
Infection type		 病情指数
Disease index
BSMV:TaPK3A 2.31** 46.2**
BSMV:GFP 1.27 25.4
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