花生丛枝植原体免疫膜蛋白基因克隆及细菌双杂交诱饵质粒构建

Characterization of Immunodominant Membrane Protein Gene from Peanut Witches' Broom Phytoplasma and Bait Plasmid Construction

初步分析植原体免疫膜蛋白的结构,以Imp构建诱饵质粒,为研究植原体与寄主互作的分子机理,探讨植原体传播、侵染及在寄主内的运输方式奠定基础。根据本实验室获得的花生丛枝植原体免疫膜蛋白基因序列设计特异性引物Imp-F/Imp-R,通过PCR扩增获得花生丛枝植原体免疫膜蛋白基因,大小519 bp,编码蛋白含有172个氨基酸残基,与SPWB膜蛋白的核苷酸差异一个碱基,氨基酸序列相同。另外与WBDL膜蛋白的核苷酸和氨基酸序列同源性分别为79.8%和70.2%。对其进行系统发育树分析;初步分析imp基因编码蛋白的跨膜区和疏水区。分析结果表明:花生丛枝植原体免疫膜蛋白C端有一跨膜锚定区,N端主要为膜内亲水区,没有前导信号序列。预测花生丛枝植原体免疫膜蛋白是一类C端跨膜的植原体免疫膜蛋白。将imp基因克隆到带有λcI基因的pBT质粒上,构建诱饵载体pBT-Imp,并通过IPTG诱导表达,western blot检测和自激活检验对其进行检测。结果显示:所构建的诱饵载体pBT-Imp可用于细菌双杂交的进一步实验。

The molecular mechanisms involving insect-vector-phytoplasma-host-plant relationships were poorly understood.Our study focused on a cell-surface membrane protein of the phytoplasma named immunodominant membrane protein（Imp）.The objective of experiment was to get the basic information between the mechanism of phytoplasma-host-plant relationships.The target ORF had been amplified using primers designed based on the conserved regions of imp gene from phytoplasma.The amplified gene was analyzed for phylogenetic tree,and the encoding protein was analyzed for transmembrane region,hydrophilic and hydrophobic region in this study.The imp gene was inserted into pBT vector including λcI gene.Construction of the pBT-Imp plasmid can be used for experiment followed by conformed that imp-λcI fusing protein was expressed by IPTG induction,western blot and self-validation testing.The immunodominant membrane protein gene from peanut witches＇ broom（PnWB） phytoplasma was 519 bp in length,encoding a predicted protein of 172 amino acids.Homology analysis of sequence from PnWB and 17 phytoplasma showed that imp gene of PnWB phytoplasma closely related to SPWB and WBDL phytoplasma,with homology rate of nucleotide being 99.9% and 79.8%,amino acids sequence,being 100% and 70.2%.Analysis of protein structure showed that the protein from PnWB phytoplasma possesses a region with C-terminal hydrophobic transmembrane anchor and N-terminal hydrophilic characterization,possibly internal to the phytoplasma cell.