小金蝠蛾酚氧化酶原基因的克隆及表达分析

Cloning and Tissue Expression Analysis of Prophenoloxidase Gene from Thitarodes xiaojinensis

通过逆转录PCR技术克隆获得小金蝠蛾(Thitarodes xiaojinensis)幼虫酚氧化酶原(TxPPO)基因cDNA的部分序列,对其进行生物信息学分析及组织表达谱分析。克隆该基因cDNA所得的部分序列长度为2654 bp,其CDS全长为2 070 bp,编码的蛋白质由689个氨基酸残基组成,预测分子质量为79.31 ku、等电点为5.91,无信号肽。进一步对TxPPO氨基酸序列分析及系统发育树构建,发现该基因与其他鳞翅目昆虫酚氧化酶原-2(PPO2)聚为一支,同源性较高,相似度为62.45%~63.61%。此外,TxPPO含有与其他鳞翅目昆虫PPO相似的丝氨酸蛋白酶酶切位点、thiolester-like位点、3个保守的血蓝蛋白结构域、2个高度保守的铜离子结合位点及位于铜离子结合位点内的6个保守的组氨酸残基。通过实时荧光定量PCR及Western blot方法对PPO基因在小金蝠蛾幼虫各组织中的表达情况进行检测,RT-qPCR结果显示,该基因在小金蝠蛾幼虫的血细胞中转录水平最高,其次为头部、脂肪体、表皮、中肠组织;Western blot检测结果表明,该蛋白分布于小金蝠蛾幼虫的血浆、头部、表皮组织及中肠组织中,而未在脂肪体检测到其分布。

A 2 654 bp partial cDNA sequence of PPO from Thitarodes xiaojinensis was cloned by reverse transcription PCR. The bioinformatics analysis showed that the coding sequence(CDS) of TxPPO contains 2 070 bp of nucleotides that encode a protein consisted of 689 amino acids. The molecular mass of the deduced TxPPO is 79.31 ku,and the isoelectric point(pI) of TxPPO is 5.91. A BLASTp search and phylogenetic tree analysis showed that the deduced amino acid sequence has a high identity to the known sequences of PPO2 from other lepidopteran insects,ranging from 62.45% to 63.61%. Besides that,the deduced TxPPO contains one proteolytic activation site,one thiolester-like site,three conserved hemocyanin domains,and two putative copper-binding sites with six conserved histidine residues,which is similar to other lepidopteran insects PPOs. Moreover,the gene expression patterns were also determined by real-time quantitative PCR and Western blot. RT-qPCR results showed that the transcription level of TxPPO was highest in hemocytes of T.xiaojinensis,followed by the head,fat body,epidermis,and midgut. Western blot results demonstrated that TxPPO was distributed in the hemolymph,head,epidermal,and midgut tissues of the larvae,but not in the fat body. In conclusion,our findings not only provide the basis to the further study on the structure and protein distribution of the PPO gene in T. xiaojinensis,but also provide a basis for further study on its function of the PPO gene.