论文部分内容阅读
【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。
【Objective】 In order to identify the expression and protein function of the tRNA-ipt gene of phytoplasma tRNA, the pathogenic mechanism of phytoplasma was explored. 【Method】 The full-length sequence of tRNA-ipt gene from the branches of Paulownia involucrata, mulberry atrophy, vinca mairei, and Melia azediostrina were analyzed by PCR and bioinformatics analysis. Prokaryotic expression of tRNA-ipt gene from Pamput flora and its preparation. Western blot and FITC indirect immunofluorescence microscopy were used to detect the expression in phytoplasma. The effect of the gene on the growth of Escherichia coli was analyzed by spectrophotometer and the cytokinin content of the transformed strain was determined by ELISA. 【Result】 The complete tRNA-ipt gene was found for the first time among Paulownia involucrata, Morus alba, Vitex negundo, and Melia azedarach. The full-length tRNA-ipt gene was 876 bp in length encoding 291 amino acids and contained ATP / GTP binding site Conserved sequence (GPTASGKT). The similarity of the amino acid sequence of tRNA-IPT between the four species was 99.1% -99.5%, which was 95.4% -99.3% homologous to the same group of phytoplasmas and less than 70% homologous to the other groups. SDS-PAGE showed that tRNA-IPT protein was expressed in E. coli. The tRNA-IPT antibody was obtained from the paulownia witches’ broom’s paratyphus for the first time and the specific expression of this protein was detected in the paulownia tissues. After the growth curve of transformed strains and the determination of zeatin content, it was found that the gene can promote the growth of Escherichia coli and the accumulation of zeatin riboside. 【Conclusion】 The tRNA-IPT protein of four plant pathogens, which encodes the same characteristics, can be expressed in phytoplasma. According to the effect of this gene on the growth rate and hormone synthesis of heterologous strains Proteins may participate in the cytokinin synthesis of phytoplasmas and play an important role in pathogenicity.