大麦中响应黄矮病毒侵染的miRNA的鉴定与功能分析

Barley yellow darft virus (BYDV) was a kind of virus that transmitted by cereal aphids and infected cereal crops seriously. It was not clear, by now, how BYDV and cereal crops interacted with each other and the resistance breeding was not proceeded effectively. It was found that miRNA had the function of resisting of viruses recently. This new finding was useful to research the mechanism between barley and BYDV and explore a new way to resisting BYDV. In this project, comparative miRNAomics is used to select and identify miRNAs responding to BYDV. At first, we isolate miRNAs from resistant and susceptible barley infected by BYDV. Then, solexa sequence technology is used to sequence miRNAs sequence. And then, different miRNAs between resistant and susceptible barley are selected and identified. At last, we use miRNA target predict and 5'-RACE analysis to identify the targets of miRNA and to analyze the function of miRNA. Based on the expression profiles of miRNAs and their targets, we analyze the difference of miRNAs between resistant and susceptible barley, between infected and uninfected barley by BYDV, to anlyze the genetic mechanism that barley respond to BYDV. The results is important to explore the molecular mechanism between BYDV and barley, and to carry out resistance breeding in Barley.

大麦黄矮病毒（BYDV）是由蚜虫传播的严重危害麦类作物的病毒。目前，BYDV与寄主间的作用机理仍不清楚，抗病育种也徘徊不前。近年来，研究发现miRNA具有抗病毒功能。这为探讨大麦与BYDV间互作机制以及抗病育种提供了新思路。本项目拟采用比较miRNA组学研究方法，分离经BYDV侵染诱导的大麦中的miRNA，采用高通量Solexa测序技术测定miRNA序列，筛选与鉴定出染毒与未染毒大麦间差异表达的miRNA。采用靶基因预测与5′-RACE分析相结合的方法，鉴定miRNA的靶基因，并分析miRNA的调控功能。基于miRNA与靶基因的表达谱，比较染毒与未染毒大麦间以及抗感病大麦间miRNA与靶基因的表达差异，筛选出响应BYDV侵染的miRNA以及与抗病相关的miRNA，探讨大麦响应BYDV侵染的遗传应答机制。研究结果将对探讨BYDV-大麦互作分子机制和开展大麦抗病育种具有重要意义。

大麦黄矮病毒（BYDV）是由蚜虫传播的严重危害麦类作物的病毒。近年来研究发现，寄主植物中的miRNA在应对病毒侵染中起到了重要的调控作用。本项目从miRNA调控基因表达的新视角出发，首先从染毒大麦（接毒蚜，BIVP）、未染毒大麦（接无毒蚜，BNVP）、正常健康对照大麦（不接蚜虫，BHCK）三个样品中分离出了小分子RNA（16-35nt），构建了对应的小RNA文库。然后采用第二代高通量测序技术Solexa与生物信息学分析相结合的策略，从这些小RNA文库中总共鉴定出160个保守的已知miRNA和312个新的候选的miRNA，并构建了这些miRNA的表达谱。通过差异表达分析，从大麦miRNA中筛选与鉴定出响应BYDV侵染而显著差异表达的miRNA有53个，其中31个上调表达，22个下调表达。这证实了BYDV的确能够诱导大麦中的miRNA差异表达。采用Northern杂交与Stem-loop qRT-PCR两种方法实验验证了这一结果的可靠性。通过靶基因生物信息学预测及5'-RACE实验验证相结合的策略，找出了响应BYDV而差异表达的大麦miRNA对应的1046个靶基因。通过对靶基因的功能注释与分析，明确了大麦中响应BYDV侵染而差异表达的miRNA在基因表达中的调控功能。采用荧光定量qRT-PCR方法建立了部分靶基因的表达谱，并结合miRNA表达谱，进一步确立了miRNA与对应靶基因之间的表达调控关系，初步探讨了在miRNA层面上大麦响应BYDV侵染的应答调控。本项目的研究结果为我们从miRNA层面上进一步探讨和理解BYDV-大麦之间互作分子机制奠定了基础。