异色瓢虫乳酸脱氢酶基因的克隆与序列分析

摘要

Lactic dehydrogenase （LDH） of Harmonia axyridis was cloned by homological cloning and anchored PCR. The full length of HaxLDH cDNA sequence is 1336 bp （GenBank accession number HM362898）, which contains a 3＇ untranslated region of 214 bp and a 5＇ untranslated region of 123 bp. HaxLDH cDNA contains an open reading frame of 999 bp, encoding a protein of 332 amino acids residues with calculated molecular mass of 36.20 kD and pI of 8.62. It has one potential N-glycosylation site （residue 352）, with no signal peptide, putative cleavage sites and transmembrane domain. Blast analysis indicated that HaxLDH contains eight conservative domains, DLQHG, TAGV/ARQK/RE/DGES/TRL, LVQRN, GSGTNLD, SCHGW/YI/VINGEHGD, VWSG/AVNV/IAGV, AYEV/IIK/RLKGYTS/NWAI/VGLS and LSLP of LDH family. HaxLDH had 67% identity with that of Tribolium castaneum, and phylogenetic analysis shows that these two species are most closely related.%乳酸脱氢酶（1acticdehydrogenase，LDH）是糖酵解途径的终止酶，在整个昆虫的发育阶段长期存在。本试验采用同源克隆和锚定PCR技术，从异色瓢虫Harmoniaaxyridis中克隆到HaxLDH基因的cDNA全序列（GenBank登录号HM362898），全长1336bp，包含214bp的3’非编码区域和123bp的5，非编码区域，可读框长999bp，编码332个氨基酸。通过软件分析，预测该基因编码蛋白的分子量为36．2kD，理论等电点为8．62；包含1个糖基化位点，无信号肽序列和跨膜结构。同源比对不同昆虫的LDH基因cDNA序列，发现HaxLDH拥有8个非常保守的结构域：DLQHG、TAGV／ARQK／RE／DGES／TRL、LVQRN、GSGTNLD、SCHGW／YI／VI／VGEHGD、VWSG／AVNV／IAGV、AYEV／IIK／RLKGYTS／NWAI／VGLS和LSLP。HaxLDH与赤拟谷盗Triboliumcastaneum的LDH同源性最高，达67％；系统发育分析也表明二者亲缘关系较近。本研究为探讨LDH在昆虫发育和逆境中的作用奠定了基础。