The Research On Defensins And Lysozymes From Aspongopus Chinensis Dallas Based On Transcriptome Sequencing And Eukaryotic Recombinant Expression

Aspongopus chinensis Dallas is a hemiptera scorpion insect.Chinese traditional medicine that is used to treat bloating,hepatogastricgia,and kidney impotence,etc.Modern medical research shows that Aspongopus chinensis Dallas have many medicinal effects such as anti-cancer,anti-bacterial,anti-inflammatory,anticoagulant,and promoting metabolism.However,there are few reports on its active substance and its mechanism of action,so it is necessary to carry out a deep and systematic study on it.In this study,118 individual individuals were collected from Guizhou province.Firstly,Molecular cloning technology.Total rna was successfully extracted from the insect lymphocytes and a cDNA library of in the lymphocytes was constructed.Two gene fragments that encoding a novel defensin DEX1 and a novel lysozyme LYM1were discovered from the transcriptome database,And the full-length cDNAs encoding DEX1 and LYM1 were successfully amplified from the constructed cDNA library.The full-length gene sequences,and the encoded amino acid sequences were analyzed by bioinformatics methods.Result of sequences alignment indicated that they showed the highest sequence similarity with defensin(79%)and lysozyme(49%)of Halyomorpha halys respectively.Secondly,according to the pPIC9K vector multiple restriction sites(EcoR I and Not I)and Pichia GS115 codon preference characteristics,the gene fragments encoding the mature peptide of DEX1 and LYM1were successfully synthesized.After the general construction steps of the eukaryotic expression vector,the recombinant pPIC9K-DEX1 and pPIC9K-LYM1 expression plasmids were obtained after PCR amplification and double enzyme digestion.Plasmids were introduced into Pichia pastoris GS115 by electroporation.PCR analysis of yeast transformants,high-copy anti-G418 selection,and Mut~+methanol use type screening were performed.Finally,a recombinant Pichia pastoris containing positive and high copy of the foreign DNA fragment of was obtained.In the present study,defensins and lysozymes were first discovered from the molecular level in the insect A.chinensis Dalls,and the recombinant Pichia pastoris expression system was constructed,which will lay a foundation for the study of the effective substance components of the insect.At the same time,it also provides ideas for the study of its innate immune system.