樟疫霉致病性相关GPCR-PIPK鉴定与机理研究

The genus Phytophthora cinnamomi causes important diseases in ornamental, agronomic and forest ecosystems worldwide. G protein coupled receptor (GPCR) and phosphatidyl inositol phosphate kinase (PIPK) are responsible for transducing extracellular signals into intracellular responses that involve complex intracellular-signaling networks, affecting the development and metabolic pathway. In this project, we predicted many putative GPCR-PIPK coding genes in the genome of P. cinnamomi according to the sequences and domains of GPCR reported in other organisms. The ortholog alleles and the gene genomic distribution patterns of the putative GPCR-PIPK encoding genes of P. cinnamomi were analyzed. The transcription profile of putative GPCR-PIPK was also performed by RNA-sequencing and qRT-PCR. The stable transformation was used to generate silenced transformants. Taken together, our results indicate essential roles of GPCR-PIPK candidate genes in development and pathogenicty in P. cinnamomi. Based on current researches, this project will further identify the downstream genes involved in GPCR-PIPK using RNA-sequcing and analyze the gene function and elucidate the regulating mechanisms. Results of this project will provide molecular targets for novel fungicide development and scientific basis for strategy of controlling Phytophthora root rot disease by P. cinnamomi.

樟疫霉引起的病害是农林生产上的严重病害，对农田、森林、园艺等经济体系造成巨大影响。G蛋白偶联受体(G protein coupled receptor, GPCR)和磷脂酰肌醇磷酸激酶（phosphatidylinositol phosphate kinase, PIPK）是两类参与不同途径的信号传导通路，对物种的生长发育阶段起着重要调控作用。本项目从樟疫霉全基因组数据库中挖掘出大量GPCR-PIPK候选编码基因，采用Real-time PCR 分析候选基因的转录表达模式，在此基础上利用瞬时沉默体系和稳定转化体系筛选出致病性相关的GPCR-PIPK，明确致病性相关GPCR-PIPK候选编码基因的生物学功能，同时鉴定其调控的下游基因的信号通路，阐明樟疫霉的生长发育和致病等重要生理过程中的作用机制，为新型杀菌剂的研发提供可能的分子靶标，从而为有效防控樟疫霉引起的病害提供理论依据。

卵菌中编码有一类新的G蛋白偶联受体，这类新型的基因在N端含有七次跨膜的GPCR结构域，C端含有磷脂酸肌醇激酶(PIPK)的结构域。樟疫霉(Phytophthora cinnamomi)中编码的GPCR-PIPK基因共有10个。本研究针对樟疫霉GPCR-PIPK基因位点，设计两条sgRNA（single guide RNA），随后将其构建入sgRNA 表达载体，从而利用CRIPSR/Cas 系统对GPCR-PIPK基因位点进行编辑，从而筛选出致病性相关新型GPCR-PIPK，明确致病性相关新型GPCR-PIPK候选编码基因的生物学功能。实验结果发现PcPIPK2(Pc212799)沉默后影响游动孢子向寄主的游动和对异黄酮的趋化性，游动孢子的休止率上升到80%，萌发率下降到50%。以上结果说明PcPIPK2在调控樟疫霉的无性发育过程中发挥作用。通过对PcPIPK2融合红色荧光蛋白进行定位分析，发现PcPIPK2定位在细胞质的小囊泡上，并且能够在细胞质中快速的移动。本研究对樟疫霉中的PcPIPK2进行了功能分析，发现PcPIPK2基因参与调控了大豆疫霉无性发育过程。后期对更多的GPCR-PIPKs基因采用新构建系统CRIPSR/Cas 系统对其进行功能研究能。GPCR-PIPKs基因在感受外界的信号、细胞质和细胞核之间物质运输和性激素的合成过程中发挥着重要的作用，敲除这些基因可导致了樟疫霉在有性生殖或者无性发育过程中产生缺陷，对于进一步理解有性生殖和无性发育的机制起着重要作用。