家蚕感染BmCPV相关未知功能蛋白LOC101742613的基因克隆及表征特征分析

Cloning and Expression Characteristics of an Uncharacterized Protein LOC101742613-coding Gene in Silkworm Related to Infection of Bombyx mori Cytoplasmic Polyhedrovirus

在感染家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrovirus,BmCPV)的家蚕中肠组织中发现一个表达量明显下调的未知蛋白LOC101742613,与棉铃虫(Helicoverpa armigera)的未知蛋白LOC110378285的序列一致性为51%。利用PCR技术克隆获得了编码该蛋白质的cDNA,其序列全长为1 114 bp,最大开放阅读框(ORF)为993 bp,编码的蛋白质由330个氨基酸残基组成,预测蛋白质分子质量为36.6 kD,等电点为4.31。实时荧光定量PCR分析发现目的基因主要在家蚕的脂肪体、精巢、卵巢和头部组织中表达,且家蚕中肠感染BmCPV后48 h和72 h其mRNA的表达量显著下调,分别为对照组的7.3%和2.1%。通过构建重组表达载体pET28a/BGIBMGA014203对去掉信号肽的目的蛋白进行原核表达,表达产物经SDS-PAGE电泳分析发现重组蛋白主要在包涵体中,分子质量约为37 kD。研究结果为进一步阐明目的基因的生物学功能奠定了基础。

A down-regulated uncharacterized protein LOC101742613 was found in Bm CPV-infected Bombyx mori midgut.This protein had 51% identity with Helicoverpa armigera uncharacterized protein LOC110378285.A 1114 bp cDNA encoding LOC101742613 was cloned by PCR.It had the longest open reading frame(ORF)of 993 bp and encoded a protein of 330 amino acids with predicted molecular weight of 36.6 kD and theoretical isoelectric point of 4.31.Fluorescent quantitative real-time PCR showed that the mRNA transcripts of target gene were mainly expressed in fat body,testicle,ovary and head.At 48 h and 72 h post-inoculation of Bm CPV,the relative expression levels of this gene in Bm CPV-infected midgut were significantly down-regulated,which were7.3% and 2.1% of that in control group.Through construction of recombinant expression vector pET28 a/BGIBMGA014203,the target protein without signal peptide sequence was expressed in E.coli.SDS-PAGE result showed that the recombinant protein with a molecular weight of 37 kD was mainly expressed in inclusion body.These results provided a basis for future study on its biological function.