低温诱导大豆疫霉游动孢子释放的基因表达谱研究及差异基因克隆

The molecular mechanism, which low temperature can promote the zoospores of Phytophthora sojae to release has not been studied. Sporangia of Phytophthora sojae induced by low temperature as materials in this project, and differential expressed gene mRNA tags induced by low temperature in sporangia will be generated by digital gene expression profiling (DGE) analysis. Additionally, high-throughput sequencing (454 GS FLX) will also applied to generate whole transcriptome profiling of the sporangia. By comparing data generated by DGE and that of 454 GS FLX, differential expressed genes and networks will be analyzed. Special expression character of the genes and signal pathways they may be involved will be revealed and confirmed by real-time quantitative PCR. Candidate differential expressed genes will be picked out based on these data, and the full length cDNAs will be synthesized by RACE technology. The results of these studies will not only revealed the molecular mechanism which low temperature can promote the zoospores of Phytophthora sojae to release, but also provide a new target sites for chemical control of Phytophthora sojae and a theoretical basis for the sustainable control of soybean blight.

目前关于低温促进大豆疫霉游动孢子释放的分子机理尚无人研究。本项目以低温处理的大豆疫霉孢子囊为研究对象，利用数字基因表达谱（digital gene expression profile，DGE）测序技术，分析经低温诱导后孢子囊的差异表达mRNA标签；同时，以高通量（454 GS FLX）测序孢子囊样品全转录组信息为对照，通过DGE 与454 GS FLX 序列数据的比对分析，获得差异基因表达谱；采用实时定量PCR 验证与分析差异表达基因的表达特征及可能参与的信号通路， 并作为候选差异表达基因，利用RACE 技术获得其基因全长cDNA。该项目的研究结果将揭示低温诱导大豆疫霉游动孢子释放的分子机理，并为药剂防治大豆疫霉提供新的靶标位点，为可持续控制大豆疫病提供理论基础。

通过低温处理大豆疫霉（Phytophthora sojae）游动孢子囊，观察了其游动孢子释放情况，证实低温有利于游动孢子的释放。为了进一步了解低温促进大豆疫霉游动孢子释放的机理，以低温处理的30min和15min的游动孢子囊为处理组，未经低温处理的为对照，采用DGE技术构建了差异文库，以FDR<0.01且差异倍数FC（Fold Change）≥2为标准筛选了差异基因。对其差异表达基因进行Blaxp注释，包括谷氧还蛋白，糖苷水解酶，锚定重复蛋白，TBP相关蛋白，乙酰转移酶，RNA指导的DNA内切酶，Flocculin, GTPase 激活蛋白，内切1，3-葡萄糖酶，钙调蛋白，磷酯酶D和磷酸-2-3-双脱氧乙醛酶。主要涉及的信号通路包括钙信号和磷酯酸信号通路。对差异基因表达进行了GO功能富集，发现低温处理15min 后，差异表达基因要涉及到结合蛋白的变化。低温处理30min后，差异表达基因涉及信号传导，核酸结构与生物合成，转录与碳的转运和代谢等方面。采用Q-PCR技术验证了差异基因表达特征。采用生物信息技术和RT-PCR克隆了与低温诱导相关的基因，包括Tyrosine-protein kinase BAZ1B ，calcium/calmodulin dependent protein kinase，phosphatidylinositol 3 and 4-kinase-like protein，phospholipase D和cellulase-3, endo-1,4-beta-glucanase基因。这结果揭示了低温促进大豆疫霉游动孢子的释放机理。