小豆抗锈病相关基因的挖掘及其表达模式分析

Adzuki bean as the traditional grain crops, which play an important role in modern agricultural industrial structure adjustment. However, adzuki bean rust occurrence has poses a threat to the sustainable and healthy development of adzuki bean production. Unfortunately, there was no efficiency disease management strategies for adzuki bean rust due to a little information known about the resistance mechanism of adzuki bean, which resulting in a decline in the quality and yield of adzuki bean. In the current study, the adzuki bean germplasm resources resistance against to the rust were evaluated, and the resistance accessions was chosen for transcriptome analysis. Resistance of adzuki bean varieties at different time points after inoculation were used for the whole transcriptome analysis, for revealing the gene expression of adzuki bean that defense against the pathogen infection. The GO and KEGG pathway enrichment analysis were conducted using the differential expressed genes. Subsequently, the differential expressed genes were certified by qRT-PCR. Genes considered involving adzuki bean resistance were chosen and the expression in different varieties with different level of resistance by using qRT-PCR. Which should clarify the target genes in the role of adzuki bean defense reaction, and lay a foundation for further study about the molecular mechanism of adzuki bean rust-resistant. The results of current study will provide a fundament for disease resistant varieties breeding and reasonable resistant varieties arrangement.

小豆作为我国传统的杂粮作物，在现代农业产业结构调整中发挥重要作用，然而，小豆锈病的严重发生已对小豆产业的持续健康发展构成了威胁。但由于对小豆的抗病机理知之甚少，对小豆-锈病菌互作规律认识粗浅，致使没有成熟的理论指导病害防治工作，导致小豆的品质和产量下降。因此，本项目拟从小豆种质资源的抗锈病鉴定入手，明确小豆资源的抗性水平，利用RNA-Seq技术分析小豆抗性品种响应锈病菌侵染的转录组，明确小豆响应病菌侵染的差异表达基因，进而采用生物信息学方法分析参与小豆抗锈病的相关信号通路及关键抗病基因，在此基础上通过定量PCR分析关键抗病基因在抗性水平不同小豆品种中的分布及其表达模式，明确抗病基因的品种特异性及其在病菌侵染不同阶段的作用。本项目对深入研究小豆抗锈病相关基因及其抗病的分子机制具有重要的理论和实践意义，为抗病品种培育及抗病基因的合理布局提供重要参考，为小豆抗病基因分子标记的开发奠定基础。

小豆作为我国传统的优势杂粮作物，在我国种植业结构调整及保障农民增收方面发挥重要作用。然而，由豇豆单胞锈菌引起的小豆锈病在我国各种植区内普遍发生，严重影响了小豆的产量和品质。因此，明确小豆资源抗锈病水平、挖掘小豆抗锈病基因、探索小豆抗锈病的分子机制，是品种创新的重要理论基础，也是实现小豆锈病绿色防控的重要依据。本项目建立了以叶片夏孢子堆数目为依据的小豆抗锈病室内评价体系，应用该体系对收集自不同地区的85份小豆资源进行了抗性评估，鉴定获得28份抗锈病小豆资源。在此基础上，选用高抗品种VaHR136，利用RNA-Seq技术对VaHR136接种后24 h和48 h的差异表达基因进行分析，共鉴定差异表达基因2348个，其中接种后24 h差异基因数目是接种后48 h的3.6倍，表明病菌侵染早期抗病品种可迅速反应进而抑制病菌的入侵。基因聚类分析发现共有137个基因在病菌侵染不同时期均被显著诱导表达，进一步的生物信息学分析表明，137个基因主要在细胞壁修饰、苯丙烷类代谢、病原识别等方面发挥重要功能，以此为依据挑选出2个感兴趣基因，N-乙酰转移酶1（VaNATA1）以及EG45结构域包含蛋白（VaEG45），通过qPCR技术分析在抗、感不同品种中锈菌侵染不同时期的表达情况，结果显示，在抗病品种中VaNATA1、VaEG45分别在病菌侵染早期（接种后12-24 h）和病菌侵染后期（接种后120 h）受病菌诱导表达量显著增高，推测这2个基因在小豆抵抗病菌入侵及扩展方面发挥重要作用。利用烟草转化体系验证VaNATA1和VaEG45在植物抗病中作用的工作目前正在进行。综合上述研究结果，本研究为明确小豆抗锈病相关基因的克隆奠定了坚实的工作基础，为小豆锈病的可持续控制提供了重要的理论依据。项目资助发表SCI论文1篇，发表会议论文3篇，待发表1篇，培养硕士研究生1名。