桑树黄化型萎缩病植原体溶血素基因MDPH的克隆及生物信息学分析和原核表达  

Cloning and Bioinformatic Analysis of the Hemolysin Gene MDPH from Mulberry Yellow Dwarf Phytoplasma and Its Prokaryotic Expression

作　　者：卢宝云[1] 韩雪娟 袁传忠[1] 李轶群 盖英萍 冀宪领[1,2] 

机构地区：[1]山东农业大学林学院,山东泰安271018 [2]怍物生物学国家重点实验室,山东泰安271018

出　　处：《蚕业科学》2012年第1期32-40,共9页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.30972366,31070573,31100478);中国博士后科学基金项目(No.20100471564);山东省优秀中青年科学家奖励基金项目(No.BS2009NY024,BS2010NY015);山东省农业良种产业化开发资助项目(No.2008167)

摘　　要：溶血素为细菌分泌的能够使细胞溶解的毒素,是病原菌重要的毒力因子。利用同源克隆技术得到桑树黄化型萎缩病植原体溶血素全长基因,命名为MDPH(GenBank登录号:HQ891118)。MDPH全长717 bp,编码238个氨基酸,预测蛋白质分子质量为27.3 kD,等电点为9.29,氨基酸序列与其它植原体中已分离的溶血素有很高的同源性。蛋白质序列结构预测表明:MDPH的二级结构中富含α-螺旋,其次为β-折叠和无规卷曲,而β-转角仅占5.46%。对蛋白质序列的理化特征预测表明:MDPH有多个亲水和疏水区域,且疏水性强于亲水性;易形成跨膜螺旋,具有7个显著跨膜结构区;蛋白的抗原性较强,不含有明显的信号肽序列,为非经典分泌蛋白。将MDPH的编码区插入原核表达载体pET30a(+),并转化到E.coli BL21中,经过IPTG诱导,MDPH在BL21菌株中成功表达。研究结果为深入探讨MDPH的功能及植原体的致病机制奠定了基础。Hemolysins are exotoxins secreted by bacteria that can lead to lysis of the host cells,being the major toxicity factor of pathogenic bacteria.A full-length gene encoding hemolysin of mulberry yellow dwarf phytoplasma was cloned and designated as MDPH（GenBank accession No.HQ891118） by homologous cloning.The gene is 717 bp long and encodes a peptide of 238 amino acids with a predicted molecular mass of 27.3 kD and an isoelectric point of 9.29.Sequence comparison analysis showed that the MDPH had high identity to hemolysins isolated from other phytoplasmas.Structure prediction to the protein sequence showed that the secondary structure of MDPH is rich in α-helixes,followed by β-strands and random coils,and only 5.46% of the amino acids forms β-turns.Prediction to physical and che-mical properties of the protein sequence indicated that MDPH possesses multiple hydrophilic and hydrophobic regions and the hydrophobicity is stronger than the hydrophilicity,being easy to form transmembrane helixes and thus leading to the formation of 7 transmembrane regions.The protein possesses evident antigenicity regions,but has no obvious peptide signal sequence,making it a non-classical secretory protein.The coding region of MDPH was inserted into the prokaryotic expression vector pET30a（＋） and transformed into Escherichia coli BL21.The gene was successfully expressed in E.coli BL2l upon induction with IPTG.The results in this study lay a good basis for further studies on functions of MDPH and pathogenic mechanism of mulberry yellow dwarf phytoplasma.

关 键 词：桑树 桑树黄化型萎缩病植原体 溶血素 基因克隆 序列结构特征 原核表达 

分 类 号：S888.712[农业科学—特种经济动物饲养] Q943.2[农业科学—畜牧兽医]