泡桐丛枝植原体tRNA异戊烯基焦磷酸转移酶基因克隆、原核表达及功能分析

Cloning， expression and characterization of tRNAisopentenyltransferase genes (tRNA-ipt) from paulownia witches'-broom phytoplasma

摘要:

摘要:【目的】为了鉴定植原体tRNA 异戊烯基焦磷酸转移酶基因(tRNA-ipt)的表达及蛋白功能，探索植原体致病机理。【方法】对泡桐丛枝、桑萎缩、长春花绿变及苦楝丛枝植原体tRNA-ipt基因完整序列进行PCR扩增和生物信息学分析。对泡桐丛枝植原体tRNA-ipt 基因进行原核表达并制备抗体。利用Western blot和FITC间接免疫荧光显微镜检测其在植原体中的表达。使用分光光度计分析该基因对大肠杆菌生长的影响，用ELISA测定转化菌株细胞分裂素含量。【结果】首次发现泡桐丛枝、桑萎缩、长春花绿变及苦楝丛枝植原体中完整tRNA-ipt基因，大小为876 bp，编码291个氨基酸，且N端均含有ATP/GTP结合位点保守序列(GPTASGKT)。4种植原体tRNA-IPT之间的氨基酸序列相似率为99.1%－ 99.5%，与同组植原体同源性在95.4%－99.3%，与其他组植原体同源性低于70%。SDS-PAGE结果显示tRNA-IPT蛋白在大肠杆菌中得到表达。首次获得泡桐丛枝植原体tRNA-IPT抗体并检测到该蛋白在泡桐发病组织中的特异表达。经过对转化菌株生长曲线及玉米素含量的测定，发现该基因能促进大肠杆菌后期生长和玉米素核苷的积累。【结论】4种植原体tRNA-ipt基因编码相同特性的功能蛋白，泡桐丛枝植原体tRNA-IPT蛋白能够在植原体中表达，根据该基因对异源菌株生长速率和激素合成的影响推断该蛋白可能参与植原体的细胞分裂素合成，在致病过程中起到重要作用。

Abstract:

Abstract:［Objective］To identify the tRNA-ipt gene of phytoplasmas and analyze the relationship between tRNA-ipt and synthesis of cytokinin as well as pathogenesis in phytoplasmas.［Methods］The paulownia witches'-broom phytoplasma (PaWB) tRNA-ipt gene was expressed in E.coli and specific antibody was prepared．Then the growth curve and cytokinin contents of E.coli with PaWB tRNA-ipt were measured by photodensitometry and ELISA respectively． ［Results］The length of tRNA-ipt genes from PaWB as well as mulberry dwarf，periwinkle virescence and Chinaberry witches'-broom phytoplasmas were 876 bp．All these genes encoded the proteins consisting of 291 amino acids．They contained and indentical motif (GPTASGKT) at N-terminal region related to the ATP or GTP binding sites．Four phytoplasma tRNAIPTs shared the 99.1－99.5%，amino acid sequence indentity with each other，shared 95.4－99.3% sequence identity with the same group phytoplasmas，whereas the less than 70% identity with 16SrX apple proliferation and 16SrXII Australia grapevine yellows phytoplasmas．The expression of the tRNA-IPT protein and localization in the phloem in phytoplasma-infected paulownia were confirmed by Western blotting and immunofluorescence detection． The determination of growth curve suggested that the growth rate increase of E.coli was affected by the transformation of exogenous tRNA-ipt gene，which might be associated with the cytokinin accumulation.［Conclusion］This protein was assumed to be involved in the cytokinin synthesis in phytoplasmas as well as other bacteria，which may play an important role in pathogenic processes of phytoplasmas and symptom development.