Acta Entomologica Sinica ›› 2020, Vol. 63 ›› Issue (6): 679-687.doi: 10.16380/j.kcxb.2020.06.03

• RESEARCH PAPERS • Previous Articles     Next Articles

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

JIANG Xue-Bing1, ZHANG Xue1, DU Wen-Mei1, ZOU Zhen2,*, ZHANG Jun-Jie1,*   

  1. (1. Engineering Research Center of Biological Control, Institute of Biological Control, Jilin Agricultural University, Changchun 130118, China; 2. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China)
  • Online:2020-06-20 Published:2020-07-02

Abstract: 【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 1 014 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.

Key words: Trichogramma dendrolimi, diapause, nitric oxide synthase, gene cloning, prokaryotic expression, qPCR