水稻细菌性褐条病菌致病相关non-coding RNAs的鉴定、功能及调控机制研究

In recent years, bacterial brown stripe is gradually becoming one of the main rice diseases in China. In order to understand the pathogenic mechanism of the pathogen, the applicant has successfully identified the composition and function of virulence-related genes such as those encoding pathogenicity islands and the type I-VI secretion systems based on the sequenced genome and transcriptome during in vivo infection of brown stripe pathogen, but little attention was paid to the role of the non-coding (nc) RNAs. Following bioinformatics analysis, a total of 353 putative ncRNAs were predicted, in which 16 ncRNAs confirmed by RT-PCR were found to be differentially expressed during in vivo infection, while there was a difference in the bacteriological characters such as pathogenicity between the constructed 4 knockout or overexpressed mutants and the wild type strain, this proposed project intends to further verify differentially expressed ncRNAs by Northern blot, to identify virulence associated ncRNAs by comparing the bacteriological characters between the wild type strain and the 12 constructed knockout and overexpressed mutants, and to eludicate the regulation mechanism of virulence related ncRNAs by determining the target gene in the term of transcriptomic comparison between the wild type and the ncRNAs mutants, and knockouting the target gene and examing the effect of mutation on the pathogenicity and expression of ncRNA. Results from this study will provide a basis for controlling brown stripe by the modulation of ncRNAs expression.

水稻细菌性褐条病这一传统次要病害近年来正逐渐上升为我国水稻主要细菌病害之一。为搞清楚该病菌的致病机理，申请者前期已基于基因组和感染转录组成功鉴定了该病菌Ⅰ-Ⅵ型分泌系统和致病岛等编码基因的组成和功能，但对非编码(non-coding, nc) RNAs则缺乏关注。本申请项目拟在生物信息学分析获得353个假定ncRNAs，半定量RT-PCR验证16个感染等条件差异表达ncRNAs，敲除或过表达其中4个影响致病性等细菌学特性的基础上；首先继续Northern杂交验证差异表达ncRNAs；然后过表达和敲除其它12个差异表达ncRNAs，比较各突变体与野生型的细菌学特性，以鉴定致病ncRNAs；最后测定致病ncRNAs突变体的转录组鉴定靶基因，敲除靶基因分析其致病功能及对ncRNAs表达的影响，以明确致病相关ncRNAs的组成及其作用机制，为通过调控ncRNAs表达来控制水稻褐条病提供基础。

鉴于水稻细菌性褐条病这一传统次要病害近年来正逐渐上升为我国水稻主要细菌病害之一，为更全面地搞清楚该病菌的致病机理以有效预防和控制该病害，本项目在前期已测定完成我国水稻细菌性褐条病菌的全基因组，保守序列生物信息学分析预测出353个假定ncRNAs的基础上，首先利用不同条件（离体、活体感染和环境细菌共培养）转录组分析获得189个表达的ncRNAs，定量PCR证实了转录组数据中的66个差异表达ncRNAs，半定量RT-PCR（或Northern杂交）最终确证了这些差异表达ncRNAs的存在；然后鉴于将近一半的假定ncRNAs位于基因内部，构建了一批差异表达ncRNAs的过表达突变体并与野生型比较，结果发现，这些差异表达ncRNAs影响褐条病菌对水稻幼苗的毒力以及其它致病相关特性，影响的程度与ncRNAs种类和参数类型有关；最后基于生信分析结合基因表达和蛋白测定，我们发现致病ncRNAs影响靶基因表达，分泌系统基因表达，效应蛋白分泌和全蛋白表达等，这一结果表明ncRNAs或许既可以直接影响水稻细菌性褐条病菌的毒力，也可以通过间接影响靶基因，分泌系统以及其它相关蛋白的表达来达到干扰植物病原细菌致病性的目的。总之，本项目已顺利完成了各项研究内容和目标，在成功鉴定水稻褐条病菌ncRNAs的基础上，进一步基本阐明了其在水稻褐条病菌致病中的功能及复杂和多元的作用机制，为通过调控ncRNAs表达来控制水稻细菌性褐条病提供了坚实的基础。