甘蓝夜蛾两种不同类型几丁质脱乙酰基酶基因的克隆与组织特异性表达

Molecular cloning and tissue-specific expression of two different chitin deacetylase cDNA sequences from Mamestra brassicae

几丁质脱乙酰基酶(chitin deacetylase,CDA)是昆虫几丁质降解酶中的一种酶,可以将几丁质转化为壳聚糖,在昆虫几丁质代谢中具有重要作用。本研究以甘蓝夜蛾Mamestra brassicae5龄幼虫虫体为材料提取总RNA,利用RT-PCR和RACE技术,分别扩增得到甘蓝夜蛾的2类不同几丁质脱乙酰基酶基因的cDNA序列。其中一种CDA含有2036个碱基,包括一个1617个碱基的开放读码框,编码一个含539个氨基酸的蛋白,分子量约为61.5ku,作者定义其为cDNA1;另一种CDA含有1312个碱基,包括一个1173个碱基的开放阅读框,编码一个含390个氨基酸的蛋白,分子量约为44.1ku,作者定义其为cDNA2。cDNA1推导的氨基酸序列具有几丁质脱乙酰基酶典型的3个功能区,分别是几丁质结合区、催化区和低密度脂蛋白受体区,属于CDA第1组的成员。cDNA2推导的氨基酸序列只有几丁质催化区,属于CDA第5组的成员。利用RT-PCR进行该基因的mRNA水平上的表达表明几丁质脱乙酰基酶cDNA1在幼虫取食期的唾腺、中肠、脂肪体组织表达,在体壁和马氏管中不表达,在预蛹期和蛹期表达;cDNA2只在幼虫期的中肠表达,在其他组织及时期都不表达。获得的基因cDNA序列已经登录GenBank并获得登录号,cDNA1登录号为HQ680620,cDNA2登录号为HQ680621。

Chitin deacetylase（CDA） is a kind of chitin-degrading enzyme found in insects that can turn chitin into chitosan and plays an important role in insects＇ chitin metabolism.Total RNA was isolated from the fifth instar larvae of Mamestra brassicae.Two CDA cDNA sequences were cloned by RT-PCR and rapid amplification of cDNA ends（RACE）.One cDNA sequence was 2 036 base pairs in length and contained an open reading frame of 1 617 base pairs coding for a polypeptide of 539 amino acid residues with a predicted molecular weight of 61.5 ku.This cDNA sequence was designated as cDNA 1.The other cDNA sequence was 1 312 base pairs in length and contained an open reading frame of 1 173 base pairs coding for a polypeptide of 390 amino acid residues with a predicted molecular weight of 44.1 ku.This cDNA sequence was designated as cDNA 2.The deduced amino acid sequence of cDNA 1 contained a polysaccharide deacetylase domain,a chitin-binding peritrophin-A domain（ChBD） and a low-density lipoprotein receptor class A domain（LDLa）.This sequence was a member of chitin deacetylase group I.The deduced amino acid sequence of cDNA 2 only contained a polysaccharide deacetylase domain.This sequence was a member of Chitin deacetylase group V.These two CDAs belong to two different CDA groups.Transcript analysis on two CDA sequences during various developmental stages and in different tissues was determined by RT-PCR.The results showed that cDNA 1 was expressed in salivary glands,the midgut and fat bodies in feeding stage,but not expressed in prepupal and pupal stage.cDNA 2 was only expressed in the midgut in feeding stage.These cDNA sequences have been deposited in Genbank with accession numbers HQ680620 for cDNA 1 and HQ680621 for cDNA 2.