异色瓢虫过氧化氢酶(Catalase)的基因克隆及序列分析

Cloning and sequence analysis of the Harmonia axyridis(Coleoptera:Coccinellinae) catalase gene

【目的】克隆异色瓢虫Harmonia axyridis保护酶系中过氧化氢酶(Catalase,CAT)的全长cDNA序列,并分析该基因的基本特性。【方法】采用同源克隆和锚定PCR技术,从异色瓢虫中克隆到HaraxCAT基因的cDNA全序列(GenBank登录号KC991026),并采用生物信息学的相关方法进行了分析。【结果】HaraxCAT的cDNA序列全长1 781 bp,其包含110 bp的3′非编码区域和45 bp的5′非编码区域,可读框长1 626 bp,编码541个氨基酸。预测该基因编码蛋白的分子量为61.55 ku,理论等电点为8.33,包含3个糖基化位点,无信号肽序列和跨膜结构。并且该基因包含了一个长达18个氨基酸的潜在的活性位点序列FDRERIPERVVHAKGAGA和血红素配体信号序列RIFSYGDTH。同源比对不同昆虫的CAT蛋白序列,发现昆虫CAT非常保守,HaraxCAT与其他昆虫的同源性高达65%及以上,与赤拟谷盗Tribolium castaneum同源性最高,达75.25%;系统发育分析表明其与鞘翅目赤拟谷盗和白星金花龟Protaetia brevitarsis亲缘关系最近。【结论】获得异色瓢虫catalase基因的cDNA全长序列,证实昆虫CAT蛋白非常保守。

[Objectives] To clone the full length cDNA sequence of the catalase gene from Harmonia axyridis, which codes for a kind of important protective insect enzyme, and analyze its basic characteristics. [Methods] Catalase （CAT） from Harmonia ax＇yridis was cloned using homological cloning and anchored PCR, and the sequence analyzed using bioinformatic methods. [Results] The full length of the HaraxCAT eDNA sequence is 1 781 bp （GenBank accession number KC991026）, which contains a 3＇ untranslated region of 110 bp and a 5＇ untranslated region of 45 bp. HaraxCAT eDNA contains an open reading frame of 1 626 bp, encoding a protein of 541 amino acids residues with a calculated molecular mass of 61.55 ku and pI of 8.33. It has three potential N-glycosylation sites, but no signal peptide, putative cleavage sites or transmembrane domain. HaraxCAT, including a proximal active site signature of FD .GAGA which encodes about 18 amino acids, also includes a proximal heme ligand signature of RIFSYGDTH. Blast analysis indicated that insect CAT proteins are very conservative; HaraxCAT has 〉 65% similarity to that of other insects. HaraxCAT had 75.25% similarity with the corresponding sequence of Tribolium castaneum, and phylogenetic analysis shows that it is most closely related to T. castaneum and Protaetia brevitarsis. [Conclusion] Sequence analysis of the catalase gene from H. aryridis confirms that insect CAT protein are very conservative.