嗜麦芽寡养单胞菌D2株Ⅱ型分泌系统GSP基因簇的序列测定及分析
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摘要
目的本实验室自沙蚕消化道分离得到的一株嗜麦芽寡养单胞菌D2可胞外大量分泌某蛋白(SMP)。根据已获知的部分基因组序列,发现该蛋白基因与Ⅱ型分泌系统蛋白基因成簇排列,故推测D2株SMP的大量分泌可能与Ⅱ型分泌系统有关。Ⅱ型分泌系统的研究为探索D2株蛋白高效分泌机制奠定基础。方法将已知的嗜麦芽寡养单胞菌D2菌株SMP基因序列通过Blast软件进行对比,发现其与嗜麦芽寡养单胞菌K279a和R551-3的AKP基因序列有高度的同源性。采用基因移步法,根据菌株K279a和R551-3的基因组序列高保守区设计上游引物,根据已获知的D2株基因组序列设计下游引物进行聚合酶链反应,将PCR扩增获得的产物均连接至p MD18-T载体中并用TSS法转化大肠杆菌JM109。利用蓝白斑筛选阳性克隆,提取质粒后经双酶切鉴定确认为阳性的菌株菌液送测序。测序所得基因片段经DNASTAR软件拼接后通过Blast比对。利用ClustalX软件分析比对后得到的完整开放读码框与GenBank相关蛋白基因序列同源性,并用MEGA3.0软件进行系统发育分析;并对各蛋白的结构进行了分析和预测。结果以嗜麦芽寡养单胞菌D2株的基因组为模板进行PCR扩增、连接、转化、酶切鉴定、测序后,分别获得7个基因片段,经拼接分析后发现其中包含了11个完整开放读码框,分别对应于II型分泌系统GspF、GspE、GspD、 GspM、GspL、GspK、GspG、GspJ、Gspl、GspH、GspC蛋白(GenBank收录号依次为：GU377212、HM151387、JQ070336、JQ070343、1Q070342、JQ070341、JQ070337、JQ070340、JQ070339、JQ070338、JQ070335)。将该11个开放读码框翻译成蛋白质后与菌株R551-3的相应蛋白序列比对和系统发育分析后发现,测序获得的11种蛋白均与同种属细菌相应蛋白亲缘关系较近。结论成功获得了嗜麦芽寡养单胞菌D2株Ⅱ型分泌系统gsp基因簇的完整基因序列,证明D2株gsp基因簇与smp紧密串联,且具有高度保守性,为进一步探索和开发利用D2株SMP蛋白高效胞外分泌表达机制奠定了基础。
Objective We obtained an protein-hyperproducing Stenotrophomonas maltophilia strain D2from the digestive tract of Perinereis aibuhitensis Grube. Based on the sequenced genes, we found the gene of this protein was clustered by genes of general secretory pathway proteins. So we speculated the type Ⅱ secretion system may be responsible for the the hyperproducion of SMP in D2. We expect to know more about the high level protein secretion mechanism of D2according to exploring the type II machinery. Methods The genes of SMP was blasted and it had a high identity with AKP gene sequences of Stenotrophomonas maltophilia K279a and R551-3correspondingly was discovered. The forward primers were designed according to the high conserved regions of K279a and R551-3, the reward primers were designed based on the sequenced genes by the methord of gene moving. Then the PCR amplified products were connected to pMD18-T-vector and transformed into Escherichia coli JM109by TSS. After sceened by white and blue clonies and identificated by double enzymes cutting, the positive clonies with the genes of interest were sequenced. The obtained gene fragments were spliced and blasted. The homology of the obtained ORFs were analysed by Clustalx and the phylogenetic relationships by MEGA3.0. Results Seven gene framents were obtained and eleven ORFs were found and named GspF, GspE, GspD, GspM, GspL, GspK, GspG, GspJ, Gspl, GspH, GspC, and their GeneBank numbers were GU377211、HM151387、JQ070336、JQ070343、JQ070342、 JQ07034、JQ070337、JQ070340、JQ070339、JQ070338、JQ070335successively. The translted amino acid sequences of the former ORFs had high identity with the corresponding proteins of both Stenotrophomonas maltophilia R551-3and K279a. The phylogenetic analyse indicated the sequenced nine proteins were homologous with the correspongding proteins of the same kind bacteria. Conclusion We obtain the complete primary structure of general secretory pathway gene cluster. The results of homologous and phylogenetic analysis lay the foundation for exploiting protein-hyperproducing mechanism of D2.
Objective We obtained an protein-hyperproducing Stenotrophomonas maltophilia strain D2from the digestive tract of Perinereis aibuhitensis Grube. Based on the sequenced genes, we found the gene of this protein was clustered by genes of general secretory pathway proteins. So we speculated the type Ⅱ secretion system may be responsible for the the hyperproducion of SMP in D2. We expect to know more about the high level protein secretion mechanism of D2according to exploring the type II machinery. Methods The genes of SMP was blasted and it had a high identity with AKP gene sequences of Stenotrophomonas maltophilia K279a and R551-3correspondingly was discovered. The forward primers were designed according to the high conserved regions of K279a and R551-3, the reward primers were designed based on the sequenced genes by the methord of gene moving. Then the PCR amplified products were connected to pMD18-T-vector and transformed into Escherichia coli JM109by TSS. After sceened by white and blue clonies and identificated by double enzymes cutting, the positive clonies with the genes of interest were sequenced. The obtained gene fragments were spliced and blasted. The homology of the obtained ORFs were analysed by Clustalx and the phylogenetic relationships by MEGA3.0. Results Seven gene framents were obtained and eleven ORFs were found and named GspF, GspE, GspD, GspM, GspL, GspK, GspG, GspJ, Gspl, GspH, GspC, and their GeneBank numbers were GU377211、HM151387、JQ070336、JQ070343、JQ070342、 JQ07034、JQ070337、JQ070340、JQ070339、JQ070338、JQ070335successively. The translted amino acid sequences of the former ORFs had high identity with the corresponding proteins of both Stenotrophomonas maltophilia R551-3and K279a. The phylogenetic analyse indicated the sequenced nine proteins were homologous with the correspongding proteins of the same kind bacteria. Conclusion We obtain the complete primary structure of general secretory pathway gene cluster. The results of homologous and phylogenetic analysis lay the foundation for exploiting protein-hyperproducing mechanism of D2.
引文
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3.刘旗,毛彩萍.嗜麦芽寡养单胞菌耐药机制的研究进展[J].实验与检验医学,2004,27(5)：517-519
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