摘要：小麦赤霉病（Fusarium Head Blight，FHB）是由禾谷镰刀菌（Fusarium graminearum）引起的小麦主要病害之一，严重影响到小麦产量和品质，对小麦抗赤霉病基因进行克隆及功能验证不仅可以解析抗赤霉病分子机理，而且有望通过基因工程加速分子育种进程。前期研究利用突变体和转录组学技术解析小麦抗赤霉病机制，筛选了一个受体类蛋白激酶基因TaRLK3-3B作为抗病候选基因。本研究利用病毒介导的基因沉默（VIGS）体系，在小麦开花期沉默TaRLK3-3B在穗部的表达，然后接种赤霉菌评价其抗性的表型变化，从而快速初步验证该基因的功能。结果显示，成功构建了TaRLK3-3B片段反向插入的载体；将该载体涂抹到小麦穗部，可成功下调TaRLK3-3B基因的表达沉默，效率达80%以上；接种赤霉菌F0609，与空白对照，平均病小穗率均提高了15%以上，平均病穗轴率提高了40%以上，植株感病性有显著提高。初步表明TaRLK3-3B具有赤霉病抗性功能。研究结果有望为抗赤霉病分子育种提供新的抗性基因资源。

关键词：小麦赤霉病；VIGS；TaRLK3-3B；抗病

Validation of the Gene Function for TaRLK3-3B in resistance to Wheat Fusarium Head Blight by VIGS system

Abstract： Fusarium head blight (FHB) is one of the major wheat diseases caused by Fusarium graminearum, which seriously affects the yield and quality of wheat. Cloning and functional verification of wheat scab genes are the prerequisite for elucidating the molecular mechanism of resistance to scab, as well as promoting molecular breeding through genetic engineering. In the previous study, a receptor-like protein kinase gene TaRLK3-3B was selected as a candidate gene for resistance to FHB by mutant induction and transcriptomics analysis. In this study, to verify the gene function of the gene, the virus-mediated gene silencing (VIGS) system was used to silence the expression of TaRLK3-3B in the panicle of wheat spike during wheat flowering, and then inoculated with FHB pathogen to evaluate the changes for its resistance. The results showed that TaRLK3-3B fragment was successfully constructed into the vector which was reversely inserted. TaRLK3-3B gene was successfully silenced in the panicle of wheat, with an efficiency of down-regulation more than 80%. Inoculation of pathogen F0609 was compared with the blank control. The average disease spikelet rate increased by more than 15%, and the average sickness rachis rate increased by more than 40%, and the plant susceptibility was significantly improved. The result showed that TaRLK3-3B play positive role in scab resistance. The research is expected to provide new resistance gene resources for molecular breeding for scab resistance.

Key words: Fusarium head blight; VIGS; TaRLK3-3B; resistance

目  录

摘要1

关键词1

Abstract…1

Key words1

引言…1

1 材料与方法…2

1.1 材料 …2

1.1.1 植物材料2

1.1.2 菌株和线性载体2

1.1.3 引物序列…2

1.1.4 实验试剂…2

1.2 实验方法2

1.2.1 引物设计…3

1.2.2 载体构建…3

1.2.3 反向插入克隆的筛选3

1.2.4 载体的线性化…3

1.2.5 体外转录反应…3

1.2.6 接种病毒…3

1.2.7 目的基因沉默效率分析…3

1.2.8 赤霉病鉴定3

2 结果与分析…4

2.1 载体构建…4

2.2 载体线性化4

2.3 体外转录…4

2.4 病毒表型…5

2.5 基因沉默效率分析…5

2.6 赤霉病鉴定5

3 讨论 6

致谢…7

参考文献8

利用VIGS体系研究TaRLK3-3B基因在小麦抗赤霉病中的功能