联合PCR技术克隆白纹伊蚊漆酶型酚氧化酶基因及其生物信息学分析

Molecular cloning and bioinformatics analysis of the whole sequence of laccase-type phenoloxidase in Aedes albopictus

目的测定白纹伊蚊漆酶型酚氧化酶的基因的全长序列并用生物信息学方法分析其结构和功能。方法从白纹伊蚊卵、幼虫、蛹、雌蚊、雄蚊中提取总RNA,逆转录后以总cDNA为模板,设计简并引物进行巢式PCR,扩增部分序列后设计新的特异性引物补全5’端序列,使用3’-RACE法补全3’端序列,拼接全长后进一步进行生物信息学分析。结果通过巢式PCR扩增出1145bp的核苷酸序列,测序后设计5’端序列的下游引物,扩增出约900bp的核苷酸序列,设计3’端的上游引物,配合3’RACE试剂盒扩增出约600bp的核苷酸序列,寻找重复序列将三段序列进行拼接,得到国内外从未获得的长2244bp、编码747个氨基酸序列的白纹伊蚊漆酶型酚氧化酶基因的全长序列。生物信息学分析其与白纹伊蚊的相似性最高,为含信号肽的跨膜蛋白,含3个Cu氧化酶超家族位点,属于蓝色多铜氧化酶家族。结论联合PCR技术的应用使较长长度的基因序列的扩增更为方便、准确。为进一步对其进行功能的研究奠定了基础。

Objective To perform molecular cloning and bioinformatics analysis of the whole sequence of laccase-type phenoloxidase（Lac） from Aedes albopictus.Methods Total RNA from eggs,larvae,pupa,female and male adult mosquitoes were prepared.Total cDNA was used as template in the design of degenerate primers for nested PCR.Specific primers were designed for partial amplification and new specific primers were used to complete 5＇ sequencing.e,use rapid amplification of cDNA ends（RACE）complete 3 ＇sequence,join together and do a further bioinformatics analysis.Results After splicing get the cDNA about 2 400 bp in length and contained an open reading frame（ORF） of 2 244 bp which encoded 747 amino acid redidues.The deduced amino acid sequence showed a high degree of identity to the reported sequence of Lac from other Lepidopterous insects.The sequence also shared the typical structural features of Lac from other insects.Bioinformatics analysis shows its a membrane protein with the signal peptide,including three Cu-oxidase super family sites,belongs to the blue many copper oxidase family.Conclusion The applications of combined PCR method make the amplification of long gene sequences more convenient and accurate.This method lays the foundation for the further reseach on the function of the gene.