橡胶树胶孢炭疽菌CgPBS2基因的克隆与功能分析

Collectotrichum leaf disease of rubber tree caused by Colletotrichum gloeosporioides(Penz) Sac. is one of the major causes of declining yields of rubber tree(Hever brasiliensis Muell.Arg.) in most rubber plantations. Despite the fact that C. gloeosporioides is ubiquitous pathogen with significant destructive potential in rubber tree in most rubber growing countries, little is known about the fungal molecular characters that are important for pathogenicity and fungicidal resistance of it on rubber tree. The high osmolarity glycerol(HOG) pathway mediates responses to hyperosmotic stress, pheromone response and virulence/pathogenicity in varous fungal pathogens. There is still no research paper published about the HOG pathway of C. gloeosporioides. In this project, we design to clone and functional analysis of CgPBS2 (PBS2-like) gene, the key component of the Hog1 pathway,in C. gloeosporioides in rubber tree. We will get the genomic DNA sequence completely based on the genome sequence of C.gloeosporioides and the flanking sequence of T-DNA inserted of mutant Cg2512(which T-DNA was inserted the promoter region of Cg PBS2), and then obtain a full-length cDNA sequence by standard RT-PCR and RACE. CgPBS2 gene deletion mutants, high expression mutants and complementary strains will be generated by gene knockout techniques and genetic complementation test. Functional analysis of CgPBS2 by observing phenotype of these mutants comparatively, and testing the relationship between the phenotype varieties and the quantity of the CgPBS2 transcripts by real-time-quantitative PCR(Q-RT-PCR) will be finished. And we also will determine the subcellular localization of CgPBS2 by fusing GFP protein to CgPBS2. After this work finished, functional analysis the CgPBS2 gene should help deciphering the determinism of stress response (such as fungicide, hyperosmotic shock, and so on.) or virulence of this important pathogen, which would be contributed to develop a new effective control of Colletotrichum leaf disease of rubber tree.

由胶孢炭疽菌侵染引起的橡胶树炭疽病是橡胶生产上最为严重的叶部病害之一。国内外对该病原菌分子机理研究报道极少。Hog1 MAPK（High osmolarity glycerol response,HOG）途径在诱导胁迫反应、信息素反应及致病性等方面都起着重要作用，但胶孢炭疽菌中对该途径的研究尚未见报道。本项目在已获得Hog1 MAPK信号途径中CgPBS2(类PBS2基因)T-DNA插入突变体的基础上，拟对该基因进行克隆与功能研究；利用基因敲除和遗传互补技术，构建基因敲除突变株、高表达突变株和互补菌株，分析表型变异情况；并利用qRT-PCR技术分析基因表达量和表型关系；同时利用GFP标签进行CgPBS2基因亚细胞定位研究。深入了解橡胶树胶孢炭疽菌Hog1 MAPK途径中CgPBS2基因的功能，这将为阐明该基因在胶孢炭疽菌适应环境胁迫的调节机制，以及橡胶树炭疽病防治策略的制定提供理论依据。

由胶孢炭疽菌侵染引起的橡胶树炭疽病是橡胶生产上最为严重的叶部病害之一。国内外对该病原菌分子机理研究报道极少。Hog1 MAPK（High osmolarity glycerol response,HOG）途径在诱导胁迫反应、信息素反应及致病性等方面都起着重要作用，但胶孢炭疽菌中对该途径的研究尚未见报道。本研究开展了橡胶树胶孢炭疽菌CgPBS2基因功能研究。在项目组前期完成该病原菌全基因组测序基础上，克隆和测序分析了橡胶树胶孢炭疽菌CgPBS2基因序列(GenBank accession number: KT387309)，采用PCR Split-Marker方法，优化转化方案（调整筛选培养基为低糖培养基），构建获得橡胶树胶孢炭疽菌CgPBS2基因缺失突变体、互补转化株、及带CgPBS2-GFP标签菌株，采用转录组测序技术，分析了野生型菌株和突变株△CgPBS2-7的基因差异表达情况。研究结果显示， CgPBS2蛋白在胶孢炭疽菌中主要分布于菌体细胞质中，但菌体细胞所处溶液渗透压会影响该蛋白的分布位置；CgPBS2基因影响胶孢炭疽菌盐胁迫功能、参与调节胶孢炭疽菌抵御渗透压功能、影响菌株对杀菌剂 fludioxonil 的抗性。获得野生型与基因缺失突变体在含和不含杀菌剂50µg/L fludioxonil的条件下转录组表达信息，初步了解CgPBS2基因调节盐胁迫、渗透压及抗药性的作用机理。在但致病功能上， CgPBS2基因并不直接影响菌株致病性，而是会影响到其他与致病性关系密切的MAPK基因表达，从而可能在一定程度上影响致病能力。