黄粉甲抗冻蛋白基因克隆及生物信息学分析

Cloning and bioinformatics analysis of antifreeze protein from Tenebrio molitor

目的: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cDNA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.采用MEGA4.0,BioEdit5.0.6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及进化分析.结果:测序结果afpTx的cDNA长度为336bp;编码112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条黄粉甲抗冻蛋白的氨基酸序列平均一致性为88%;与11条赤翅甲抗冻蛋白氨基酸序列的平均一致性为67%,2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.

AIM：To obtain sequence coding gene for the antifreeze proteins（AFP）from local Tenebrio molitor and to elucidate the 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 analyzedby MEGA 4.0 and BioEdit 5.0.6 computer program for amino acid sequence homology and evolutionary variance.RESULTS：Sequencing result showed a correctly constructed vector that containing 336 bp antifreeze protein cDNA.Digestion and electrophoresisresults confirmed that gene was successfully cloned and subcloned into pET32a（＋）.Sequence similarity analysis indicatedthat our afpTx sequence showed a sequence homology of 88% to 23 GenBank submitted unique sequences of Tenebrio molitor AFPs,and an average homology of 67% to 11 Dendroides Canadensis AFPs and 63% to two beetle species.Phylogenetic analysis indicated that two beetle species are homologous and striking feature of this analysis showed that DNA sequence divergence among Dendroides Canadensis were markedly higher that among Tenebrio molitor.CONCLUSION：Successfully cloned local afpTx sequenceand proved that this sequence is homologous among two beetle isoforms.