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【目的】Pbs2是MAPK信号途径HOG-MAPK通路的重要成员之一,在植物病原菌渗透压调节方面发挥着重要作用。橡胶树白粉病菌(Oidium heveae)是专性寄生菌,论文借助橡胶树炭疽病菌(Colletotrichum gloeosporioides),研究橡胶树白粉病菌OhPbs2的功能。【方法】采用同源克隆方法,分别以橡胶树白粉病菌基因组DNA和c DNA为模板,PCR扩增OhPbs2;利用生物信息学分析该基因的结构域,对该基因和其他真菌的7个同源蛋白序列进行系统进化分析,并利用MEGA6中最大简约法构建系统进化树来进一步分析鉴定该基因;利用同源重组和原生质体转化技术,将OhPbs2转化到缺失Pbs2的橡胶树炭疽菌突变体ΔCgPbs2中;在PDA+1.5 mol·L-1山梨醇的平板上筛选转化子,同时提取转化子的基因组作为模板,用橡胶树白粉病菌OhPbs2的引物对进行PCR鉴定,选择正确的转化子ΔCgPbs2+OhPbs2进行后续表型测定;在不同培养条件下,比较ΔCgPbs2、ΔCgPbs2+OhPbs2和野生型菌株生长状态,并在橡胶树叶片接菌检测3个菌株致病力。【结果】OhPbs2基因序列全长1 927 bp,c DNA序列全长1 860 bp,含有一个内含子,编码一个619个氨基酸的蛋白;生物信息学分析表明该蛋白具有与Cg Pbs2相同的S_TKc功能域,构建系统进化树发现橡胶树白粉病菌Pbs2蛋白与烟曲霉(Aspergillus fumigatus)Pbs2蛋白亲缘关系最近,相似度为55%,与橡胶树炭疽病菌的相似度为49%,与粗糙脉孢菌(Neurospora crassa)、稻瘟菌(Magnaporthe oryzae)、酿酒酵母(Saccharomyces cerevisiae)、禾谷镰孢(Fusarium graminearum)的Pbs2蛋白具有较近的亲缘关系,相似度分别为54%、53%、53%、50%;ΔCgPbs2+OhPbs2菌株能在PDA+1.5 mol·L-1山梨醇培养基上长出白色菌落,ΔCgPbs2菌株不能生长,并且测序结果显示OhPbs2已成功转入ΔCgPbs2;ΔCgPbs2+OhPbs2在MM培养基上菌落呈白色,气生菌丝较短,不同于wild type菌株。在含有不同浓度Na Cl、山梨醇、SDS、H2O2及咯菌腈的MM培养基上,ΔCgPbs2、ΔCgPbs2+OhPbs2和wild type菌株随着浓度增加,菌落生长速度逐渐降低,OhPbs2不仅能恢复橡胶树炭疽菌菌株耐渗透压尤其耐受山梨醇的能力以及对咯菌腈的敏感性,甚至具有增强的能力,OhPbs2互补改变了橡胶树炭疽病菌颜色,抑制了气生菌丝生长;Cg Pbs2可能参与了橡胶树炭疽病菌致病性,但OhPbs2互补没有恢复其致病性。【结论】OhPbs2可能参与调控病菌的营养生长、氧化应激反应、渗透压响应及细胞壁形成等,并能增强橡胶树炭疽病菌相应功能,但与Cg Pbs2调控病菌致病力的机制可能存在不同。
【Objective】 Pbs2 is one of the important members of HOG-MAPK pathway in MAPK signaling pathway and plays an important role in the regulation of osmotic pressure in plant pathogens. The rubber plant, Oidium heveae, is an obligate parasitic organism. The paper studied the function of rubber tree powder OhPbs2 with the help of Colletotrichum gloeosporioides. 【Method】 OhPbs2 was amplified by PCR using genomic DNA and c DNA of powdery mildew of Rubber Tree, respectively. The domain of this gene was analyzed by bioinformatics analysis. The homology of 7 homologous proteins of this gene and other fungi Sequence analysis and phylogenetic tree using the largest parsimony MEGA6 to further analyze and identify the gene; using homologous recombination and protoplast transformation technology, OhPbs2 will be transformed to Pbs2 deletion of the P. meliloti mutant ΔCgPbs2; in PDA + 1.5 mol·L-1 sorbitol. The transformant was also extracted and the genome of the transformant was selected as a template. The PCR products were identified by the primers of OhPbs2, and the correct transformants ΔCgPbs2 + OhPbs2 were selected for the subsequent phenotype The growth of ΔCgPbs2, ΔCgPbs2 + OhPbs2 and wild type strains were compared under different culture conditions. The pathogenicity of three strains was tested on rubber tree leaves. 【Result】 The full-length cDNA of OhPbs2 gene was 1 927 bp in length and 1 860 bp in length. It contained an intron and encoded a protein of 619 amino acids. Bioinformatics analysis showed that this protein had the same S_TKc function as Cg Pbs2 The phylogenetic tree was constructed and phylogenetic tree was constructed. The Pbs2 protein of Aspergillus fumigatus was closest to Pbs2 protein of Aspergillus fumigatus, with a similarity of 55% and a similarity of 49% with P. melitensis, which was similar to Neurospora crassa ), Psap2 protein of Magnaporthe oryzae, Saccharomyces cerevisiae and Fusarium graminearum had similar genetic relationship with similarity of 54%, 53%, 53%, 50% ; ΔCgPbs2 + OhPbs2 strains can grow white colonies on PDA + 1.5 mol·L-1 sorbitol medium, ΔCgPbs2 strain can not grow, and the sequencing results showed that OhPbs2 has been successfully transferred ΔCgPbs2; ΔCgPbs2 + OhPbs2 colonies in MM medium White, aerial mycelium shorter, different from wild type strains. With increasing concentrations of ΔCgPbs2, ΔCgPbs2 + OhPbs2 and wild type strains on MM medium containing different concentrations of NaCl, sorbitol, SDS, H2O2 and fludioxonil, colony growth rate decreased gradually, OhPbs2 can not only restore rubber tree anthrax Strain osmotolerance is particularly resistant to sorbitol and susceptibility to fludioxonil, even with enhanced ability, OhPbs2 complementarity altered the color of R. solanacearum and inhibited the growth of aerial mycelia; Cg Pbs2 may be involved in rubber tree anthrax Pathogenic bacteria, but OhPbs2 complement did not restore its pathogenicity. 【Conclusion】 OhPbs2 may be involved in the regulation of vegetative growth, oxidative stress response, osmotic pressure response and cell wall formation of bacteria, and may enhance the corresponding function of the pathogen of rubber tree. However, the mechanism of the pathogenicity of the bacteria may be different from that of Cg Pbs2.