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
唐 斌1, 2** , 施佐堃1 , 郭红双1 , 王 甦2 ,王世贵1 , 张 帆2***
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作者单位：1. 杭州师范大学生命与环境科学学院，杭州 310036；2. 北京市农林科学院植物保护环境保护所，北京 100097
中文关键词：异色瓢虫，过氧化氢酶，基因克隆，序列分析
英文关键词：Harmonia axyridis, catalase, gene cloning, sequence analysis
中文摘要： 【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。
英文摘要： [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 axyridis was cloned using homological cloning and anchored PCR, and the sequence analyzed using bioinformatic methods. [Results] The full length of the HaraxCAT cDNA 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 cDNA 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 FDRERIPERVVHAKGAGA 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. axyridis confirms that insect CAT protein are very conservative.