黄粉甲抗冻蛋白基因克隆及生物信息学分析任谦1熊鸿燕1朱才众1.DOC

黄粉甲抗冻蛋白基因克隆及生物信息学分析 任谦1 ,熊鸿燕1,朱才众1,张世界2（1第三军医大学军事预防医学院军队流行病学教研室，重庆 400038；2西藏军区山南分区后勤部卫生科，山南地区 850700） 获得黄粉甲抗冻蛋白基因afpTx及相关生物信息学资料。[方法]从黄粉甲幼虫中提取总RNA，通过RT-PCR合成黄粉甲抗冻蛋白基因afpTx的成熟肽片段，克隆入载体pMD19-T,进行测序分析；酶切后将其亚克隆入表达栽体pET32a（+），构建表达质粒pET32a-afpTx,并转化到大肠杆菌DL21后提取质粒，双酶切鉴定。用MEGA 4.0，BioEdit 5.0.6软件对本研究克隆的抗冻蛋白基因afpTx进行氨基酸序列同源性变异和进化分析。[结果]测序结果afpTx的成熟肽长度为bp；编码84个氨基酸；酶切、电泳结果表明克隆和亚克隆获得成功。抗冻蛋白氨基酸序列相似性分析表明afpTx与GenBank上提交的23条黄粉甲同种异构体的氨基酸序列平均一致性为88%；与11条赤翅甲氨基酸序列的平均一致性为67%；两种甲虫的平均一致性为63%。进化树分析结果显示黄粉甲与赤翅甲抗冻蛋白序列是同源序列。赤翅甲的序列趋异度显著大于黄粉甲抗冻蛋白基因序列。[结论] 成功克隆了本地黄粉甲的afpTx基因该序列是GenBank上提交的黄粉甲与赤翅甲抗冻蛋白的同源序列。【关键词】黄粉甲;抗冻蛋白；克隆、afpTx基因;生物信息学Cloning and Bioinformatics analysis of Antifreeze Protein From Tenebrio molitor Ren Qian1, Hongyan Xiong1 , Caizhong Zhu1,Shijian Zhang2 (1 Department of Epidemiology, 3rd Military Medical University, Chongqing,PR China University, Gao Tan Yan Zheng Jie,no.30,Sha Ping Ba District,Chongqing 400038,China, 2 Shan Nan Military Sub-District Health Branch Shan Nan District 850700 China.) 【Abstract】Objective: To obtain sequence coding for the antifreeze proteins(AFP) from local Tenebrio molitor by gene engineered and study related bioinformatics data. METHODS: After the total RNA was isolated, from the larva of Tenebrio molitor. cDNA encoding the afpTx was synthesized by RT-PCR, and the PCR products were inserted into the vector pMD19-T simple, which were subcloned into pET-32a ( + ) and transformed into E. coli and identified with restriction enzyme analysis. Then the sequencing result was analyzed by MEGA 4.0 and BioEdit 5.0.6 computer program, with which aligned amino acid sequence of afpTx homology and evolutionary variance. RESULTS: Sequenced successfully, Mature peptide coding is 252bp and encode 84 amino acids. Digestion and electrophoresis results showed that the success of cloning and subcloning. Antifreeze protein amino acid sequence similarity analysis showed that the afpTx and 23 Genbank submitted unique sequence of Tenebrio molitor AFPs identities an averag