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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), an enzyme in insect chitin degrading enzymes, can convert chitin into chitosan, which plays an important role in insect chitin metabolism. In this study, total RNA was extracted from the fifth instar larvae of Mamestra brassicae larvae. The cDNA sequences of two different chitin deacetylase genes were amplified by RT-PCR and RACE respectively. One of the CDA contains 2036 bases, including a 1617 base open reading frame encoding a 539 amino acid protein with a molecular weight of about 61.5ku, the authors define it as cDNA1; the other CDA contains 1312 The base, which contains an open reading frame of 1173 bases, encodes a protein of 390 amino acids with a molecular mass of about 44.1ku, which the authors define as cDNA2. cDNA1 deduced amino acid sequence with chitin deacetylase typical three functional areas, namely chitin binding region, catalytic region and low density lipoprotein receptor region, belonging to CDA group 1 members. cDNA2 deduced amino acid sequence only chitin catalytic region, belonging to CDA group 5 members. Expression of mRNA of this gene by RT-PCR showed that chitin deacetylase cDNA1 was expressed in salivary glands, midgut and fat body during larval feeding stage, but not in body wall and duct. Pre-pupal and pupal expression; cDNA2 only in the larval midgut expression, in other tissues and time are not expressed. The obtained gene cDNA sequence has been registered GenBank and accession number, cDNA1 accession number HQ680620, cDNA2 accession number HQ680621.