小麦-簇毛麦6DL•6V#4S易位系PM97033白粉病广谱抗性机制研究

Powdery mildew (PM) is a main factor limiting wheat production and quality. Wheat wild species is the important genetic resource for wheat improvement on this disease. Even though some disease resistance genes to PM from wild relatives transferred into wheat have showed great application potential, these resistance genes and their mechanisms have been remained largely unknown. In this program, the PM pathogen infected wheat leaves will be used for dynamic transcriptome sequencing of both host and pathogen in their early interaction, by employing a 6DL•6V#4S translocation line PM97033 with strong PM resistance, which was derived from a cross between wheat and Dasypyrum villosum originated from the former Soviet Union, and its susceptible wheat parent as materials. We will comprehensively apply bioinformatics and molecular biology strategies to find candidate resistance genes in the host and pathogenicity factors in the pathogen, which are differentially expressed between incompatible and compatible interaction groups, respectively. By using differential gene screening, collinearity analysis, PCR amplification, and sequencing confirmation, the unigenes from 6V#4S will be identified. In virtue of gene annotation, the effector protein coding genes expressed differentially in the pathogen will be screened. Expression models of candidate genes in the time course of the interaction will be analyzed by qRT-PCR. Full length of the genes will be isolated by some techniques like RACE. Functions of candidate resistance genes to PM will be fast evaluated using single-cell transient overexpression and VIGS. The PM resistance of some key candidate genes will be tested in wheat by Agrobacterim-mediated stable transformation. The results of this project will lay foundation for elucidating the molecular mechanism of PM resistance in PM97033.

白粉病是小麦产量和品质的主要限制因素之一，一些近缘植物的抗病性在育种上具有巨大应用潜力，但其抗病基因及作用机制尚缺乏研究。本项目以利用前苏联簇毛麦衍生的6DL•6V#4S抗白粉病易位系PM97033及其小麦亲本为材料，采用白粉菌侵染的小麦叶片进行动态性寄主-病原菌转录组测序；综合应用生物信息学、分子生物学方法，在亲和互作与非亲和互作间的差异表达基因中寻找候选的寄主抗病基因及病菌毒性因子；通过差异基因筛选、共线性分析、PCR扩增及测序验证等方法，鉴定来自6V#4S的抗病相关基因；借助基因注释筛选白粉菌差异表达的效应子蛋白基因；利用qRT-PCR分析候选基因表达模式，RACE等技术克隆候选基因全长，单细胞瞬间过表达和VIGS快速鉴定候选基因功能，转基因技术鉴定关键候选基因的白粉病抗性。本研究将为阐明PM97033抗病的分子机制奠定基础。

小麦-簇毛麦6DL·6V#4S易位系Pm97033抗白粉病，与6AL·6V#2S易位系中已知的抗白粉病基因Pm21具有相同的同源群，但2条不同来源的6VS遗传基础是否相同仍不明确。本研究利用Pm97033及其感病背景亲本宛7107为材料，对白粉病菌侵染过程中寄主-病菌进行双向转录组测序，应用生物信息学、分子生物学方法筛选和鉴定6V#4S上与白粉病抗性相关的基因，利用VIGS、转基因技术和基因编辑进行功能鉴定。结果表明，从Pm97033相比宛7107经白粉菌侵染的转录组无参数据中筛选到220个ungenes，将35个unigenes定位于6V#4S染色体上，其中的15个unigenes与抗病基因相关（7个unigenes受白粉菌诱导上调表达）。这些unigenes可以作为特异鉴定6VS以及6V#4S与6V#2S的分子标记，大部分unigenes在小麦第6部分同源群和大麦6H短臂上具有较好的共线性。进一步从Pm97033相比宛7107经白粉菌侵染的转录组无参数据中获得了一定数量的差异表达基因，12h和48h之间有566个重叠基因，P12相比W12和P48相比W48都表达的抗病相关基因有22个，其中，c112345_g2和c112345_g3注释到Pm21。从白粉菌转录组393个注释为效应子蛋白的基因中筛选到在亲和互作中表达量较高的15个基因，进一步筛选到3个在亲和与非亲和互作中差异表达的基因和1个功能注释为糖基水解酶的基因，它们的瞬时表达未观察到明显的毒性表型。抗病相关基因DvLox、DvFbx和Pm21#4在Pm97033中沉默之后都影响到了其对白粉菌的抗性，过表达DvLox的植株对白粉菌具有一定抗性，过表达Pm21#4的植株对白粉菌免疫，基因编辑敲出Pm21#4植株对白粉菌高度感染，过表达Pm21#4-H基因同样对白粉菌免疫，Pm21#4-H属于一个新基因，但不受白粉菌诱导表达。本研究揭示了易位系Pm97033白粉病广谱抗性的遗传基础，对小麦抗白粉病育种具有很好参考价值。