菜青虫胰凝乳蛋白酶基因PrCT1的克隆及表达分析

Cloning and Expression Analysis of Chymotrypsin Gene PrCT1 of Pieris rapae

胰凝乳蛋白酶是昆虫的主要消化酶,可能参与多种消化以外的生理过程。为了解该蛋白质基因表达特性,基于菜青虫(Pieris rapae)转录组数据,克隆了1个菜青虫胰凝乳蛋白酶基因PrCT1并进行相关分析。该基因编码区全长861 bp,编码286个氨基酸,含有典型的丝氨酸蛋白酶催化三联体(H57、D102、S195),经丝氨酸蛋白酶底物特异性口袋分析,显示其属于胰凝乳蛋白酶。PrCT1与黑脉金斑蝶胰凝乳蛋白酶(GenBank登录号:OWR53227)同源性最高,达到49%,且亲缘关系最近。构建pET28a-PrCT1原核表达重组载体,转化大肠杆菌Rosetta(DE3)菌株进行原核诱导表达。结果发现,重组PrCT1蛋白以包涵体形式存在,分子质量为33.26 ku。利用实时荧光定量PCR检测菜青虫不同组织中PrCT1 mRNA表达及饥饿、决明胰蛋白酶抑制剂喂食处理后PrCT1 mRNA的表达情况显示,PrCT1基因主要在菜青虫的中肠中表达,且在饥饿、决明胰蛋白酶抑制剂喂食处理后表达水平均下调,推测PrCT1可能是菜青虫中肠中发挥食物消化作用的重要蛋白酶,且可能是重要的抗虫靶点。

Chymotrypsin is the main digestive enzyme of insects and may have many other functions.In order to understand the gene expression characteristics of this protein,a chymotrypsin gene PrCT1 was cloned and analyzed based on the transcriptome data of Pieris rapae.The gene included an ORF of 861 bp and encoded a polypeptide of 286 amino acids with a typical serine protease catalytic triplet(H57,D102 and S195).PrCT1 was shown to be a member of chymotrypsin by analysis of the serine protease substrate-specific pocket.PrCT1 shared a high sequence identity(49%)and had the closest relationship with the chymotrypsin of Danaus plexippus(GenBank accession number:OWR53227).The prokaryotic recombinant vector pET28a-PrCT1 was constructed and transformed into E.coli Rosetta(DE3)strain to express the target protein.The results showed that PrCT1 protein existed as inclusion bodies with molecular mass of 33.26 ku.The expression of PrCT1 mRNA in different tissues of Pieris rapae and the impacts of starvation and Cassia obtusifolia trypsin inhibitor feeding on the expression of PrCT1 mRNA were detected by qRT-PCR.The results showed that PrCT1 gene was mainly expressed in the midgut of Pieris rapae,and the expression level was down-regulated after starvation and Cassia obtusifolia trypsin inhibitor feeding treatments.As a result,PrCT1 might be an important enzyme taking part in food digestion in the midgut of Pieris rapae,and might be an important target of insecticide.