利用酿酒酵母YPG30验证橡胶树白粉菌OhIsc1蛋白的分泌功能  

Verification of Secretory Function of Oidium heveae Oh Isc1 Protein Us ing Saccharomyces cerevisiae Strain YPG30

作　　者：石豆 刘玉涵 李思鹏 何其光[1] 刘文波[1] 缪卫国[1] 李潇[1] Shi Dou;LiuYuhan;Li Sipeng;He Qiguang;Liu Wenbo;Miao Weiguo;Li Xiao(Key Laboratory of Ministry of Education for Green Prevention and Control of Tropical Plant Diseases and Pests,College of Plant Protection,Hainan University,Haikou,570228)

机构地区：[1]海南大学植物保护学院,热带农林生物灾害绿色防控教育部重点实验室,海口570228

出　　处：《基因组学与应用生物学》2021年第4期1597-1603,共7页Genomics and Applied Biology

基　　金：海南省重点研发计划项目(ZDYF2018240);国家自然科学基金项目(31660033);国家重点研发计划项目(2018YFD0201105)共同资助。

摘　　要：橡胶树白粉菌(Oidium heveae)引起的橡胶树白粉病严重影响天然橡胶产量而造成经济损失。水杨酸(salicylic acid, SA)是植物细胞内一种重要的抗病防卫反应的信号分子。植物病原物在致病过程中会分泌出异分支酸酶(isochorismatase, ISC)水解异分支酸,从而抑制植物中SA的积累并影响植物的抗病性。本研究通过生物信息学方法鉴定到橡胶树白粉菌中存在一个异分支酸酶同源蛋白的编码基因(OhIsc1),长度为693 bp,具有3个内含子,cDNA大小为600 bp,编码199个氨基酸,且该蛋白为预测的非经典型分泌蛋白,具有ISC保守结构域,属于异分支酸酶蛋白家族,但不具有信号肽。利用同源克隆法获得OhIsc1的cDNA序列,并构建p YES2-OhIsc1载体,将载体转化到酿酒酵母(Saccharomyces cerevisiae)菌株YPG30,获得表达OhIsc1的转化子。通过Western blot免疫印迹方法在转化子的胞外液中检测到OhIsc1-HA,表明OhIsc1可被酵母细胞分泌至胞外。本研究证实OhIsc1为分泌蛋白,为后续深入研究验证OhIsc1的分泌功能及其在病菌致病过程的角色提供了基础。Rubber powdery mildew caused by Oidium heveae seriously affects the rubber production and causes economic losses. Salicylic acid(SA) is an important signal molecule for disease-resistant defense responses in plant cells. Plant pathogens secrete isochorismatase(ISC) during pathogenesis, thereby ISC can inhibit the accumulation of SA in plants and affect the disease resistance of plants. In this study, we identified a gene encoding a homologous protein(OhIsc1) with a length of 693 bp, 3 introns, and a cDNA size of 600 bp encoding 199 amino acids in O. heveae genome by bioinformatics analysis. Moreover, the protein, a predicted non-classical secreted protein,had an ISC conserved domain, belonging to a family of ISC, but had no signal peptide. The cDNA sequence of OhIsc1 was obtained by homologous cloning and the pYES2-OhIsc1 vector was constructed. S. cerevisiae strain YPG30 was transformed with pYES2-OhIsc1 vector and the transformant was obtained. OhIsc1-HA was detected in the extracellular fluid of the transformant by Western blot immunoblotting, indicating that OhIsc1 can be secreted to extracellular space by yeast cells. This study confirmed that OhIsc1 was a secreted protein, which provided a foundation for its role in the pathogenesis of pathogens.

关 键 词：橡胶树白粉菌 OhIsc1蛋白 异分支酸酶 非经典型分泌蛋白 

分 类 号：S763.7[农业科学—森林保护学]