泡桐丛枝植原体Sec分泌蛋白转运系统3个亚基基因的克隆及蛋白特性分析

Cloning and characterization of three subunits of the phytoplasma Sec protein translocation system associated with the paulownia witches'-broom

采用PCR方法扩增Sec分泌蛋白转运系统3个亚基基因,并进行了生物信息学分析。通过SWISS-MODEL同源建模与3D-PSSM折叠子识别法构建蛋白质的三维结构,并通过PROCHECK程序验证构建的三维结构的可靠性。首次从泡桐丛枝(PaWB)植原体基因组中分离出Sec分泌蛋白转运系统3个亚基基因,各基因全长依次为2 508、1 242 bp和411 bp,分别编码835、204个及136个氨基酸的蛋白。SecA、SecY和SecE均为脂溶性稳定蛋白,SecA无明显跨膜区,SecY和SecE分别含有10个和3个明显的疏水跨膜区。二级结构主要为螺旋,其次为折叠和无规则卷曲,没有转角。构建的三维结构符合立体化学原则。泡桐丛枝(PaWB)植原体中存在的Sec分泌蛋白转运系统作为最主要的运输途径,可能直接转运菌体蛋白如毒素等直接到寄主细胞质中或介体昆虫细胞中,引起寄主病症。研究植原体的Sec蛋白转运系统对于了解植原体的致病机理及预防这类病害的发生提供了理论依据。

Three subunits secA, secY and secE genes were amplified by polymerase chain reaction from infected paulownia plants, and the proteins encoded by them were analyzed by bioinformaties. The three-dimensional structures were construeted by SWISS-MODEL homology modeling and 3D-PSSM folds recognition, then analyzed and validated by the program PROCHECK. Three genes secA, secY and secE were obtained. The full length of the three genes were 2 508 bp, 1 242 bp and 411 hp in length, respectively, encoding 835, 204 and 136 amino acid residues respectively. Bioinformatie analysis of these subunits showed that SecA, SecY and SecE are all liposoluble stable protein. SecA has no transmemnrane domain, but SecY and SecE which have 10 and 3 evident hydrophobic transmembrane domains, respectively, belonging to membrane integral proteins. Their secondary structure contains mainly helix, secondly sheet and coil, hut no turn. The constructed three --dimensional structure accords with the stereochemistry after validation. The Sec protein translocation system as the most important protein translocation pathway may secrete toxins directly into host cytoplasm or the cells of insect vector, resulting in host plant infection. The study on Sec protein transloeation system might provide a theoretical basis for understanding the pathogenesis of phytoplasma and preventing the occurrence of these diseases.