松毛虫赤眼蜂一氧化氮合酶基因的克隆、原核表达及在不同滞育阶段的表达谱分析

Molecular cloning and prokaryotic expression of nitric oxide synthase gene from Trichogramma dendrolimi(Hymenoptera:Trichogrammatidae)and its expression profiling across different diapause stages

【目的】本研究旨在克隆松毛虫赤眼蜂Trichogramma dendrolimi一氧化氮合酶(NOS)基因,表达其编码蛋白,并探究其在松毛虫赤眼蜂滞育过程中的表达模式,为松毛虫赤眼蜂滞育研究提供新依据。【方法】通过转录组数据分析获得松毛虫赤眼蜂一氧化氮合酶基因TdNOS cDNA序列,使用qPCR方法检测其在松毛虫赤眼蜂不同滞育阶段预蛹和蛹中的表达量。使用RT-PCR方法扩增TdNOS cDNA序列并通过生物信息学方法分析其序列特征。构建TdNOS的原核表达载体,利用大肠杆菌Escherichia coli表达系统表达重组TdNOS蛋白,通过Ni-NTA琼脂糖亲和层析纯化,并用于制备多克隆抗体。利用Western blot进一步检测TdNOS在松毛虫赤眼蜂不同滞育阶段的表达量。通过质谱鉴定纯化的目标蛋白。【结果】qPCR分析表明,TdNOS在松毛虫赤眼蜂滞育解除后蛹期的相对表达量显著高于其他阶段的。克隆获得松毛虫赤眼蜂TdNOS cDNA序列(GenBank登录号:MN650600),开放阅读框(ORF)序列长1014 bp,编码337个氨基酸,无信号肽序列,不含跨膜结构,预测有33个丝氨酸磷酸化位点、7个酪氨酸磷酸化位点和10个苏氨酸磷酸化位点。系统发育分析表明,松毛虫赤眼蜂NOS与短管赤眼蜂T.pretiosum NOS亲缘关系最近,形成与其他膜翅目昆虫NOS蛋白分离的独立分支。成功表达纯化了重组蛋白TdNOS并制备了抗体;Western blot结果表明,TdNOS在松毛虫赤眼蜂滞育解除后蛹期的蛋白表达量高于其他阶段的。质谱检测结果表明纯化的目的蛋白即为TdNOS。【结论】松毛虫赤眼蜂TdNOS在解除滞育后蛹期具有较高的蛋白及mRNA表达量。本研究为进一步探究松毛虫赤眼蜂一氧化氮合酶在滞育发育过程中的作用机制奠定了基础。

【Aim】The aim of this study is to clone nitric oxide synthase(NOS)gene of Trichogramma dendrolimi,to express its coded protein and to investigate its expression profile during diapause in T.dendrolimi,so as to provide a new basis for the study of T.dendrolimi diapause.【Methods】The cDNA sequence of TdNOS of T.dendrolimi was obtained based on the transcriptome analysis,and its expression levels in prepupae and pupae at different diapause stages of T.dendrolimi were detected using qPCR.The cDNA sequence of TdNOS was cloned using RT-PCR,and the sequence features were analyzed by bioinformatics methods.The prokaryotic expression vector of TdNOS was constructed.The recombinant TdNOS was expressed using Escherichia coli expression system,purified using Ni-NTA agarose affinity chromatography and used to prepare polyclonal antibody.The expression levels of TdNOS at different diapause stages of T.dendrolimi were further detected using Western blot.Mass spectrometry(MS)was used to identify the purified protein.【Results】The mRNA abundance of TdNOS was significantly higher at the pupal stage after diapause than at other stages of T.dendrolimi.The cDNA sequence of TdNOS(GenBank accession number:MN650600)was obtained by PCR.Its open reading frame(ORF)is 1014 bp in length,encoding 337 amino acids without signal peptide sequence and transmembrane structure.The encoded protein was predicted to have 33 serine phosphorylation sites,seven tyrosine phosphorylation sites,and 10 threonine phosphorylation sites.Phylogenetic analysis showed that TdNOS and NOS of T.pretiosum was closely related,forming an independent clade separated from NOS proteins of other hymenopteran insects.The recombinant TdNOS was expressed and purified,and the polyclonal antibody was prepared.The Western blot results showed that the expression level of TdNOS at the pupal stage after diapause was higher than that at other stages of T.dendrolimi.The results of MS showed that the purified protein was NOS.【Conclusion】TdNOS has higher mRNA and protein expression at the pupal stage after diapause.This study lays a foundation for further exploring the role of TdNOS in regulating diapause.