Molecular Cloning,Characterization And Expression Analysis Of The Defensin Gene CcDef From Coridius Chinensis

Antibiotics are the most widely used drugs.The emergence and spread of antibiotic-resistant strains increase the difficulty in the treatment of some infectious diseases.Insect antibacterial peptide is a peptide compound with immune function,and expected to replace antibiotics as a new type of antibacterial drug due to its special antibacterial mechanism.In the animal world,insect is the largest biological group in Animalia and is found everywhere in the world.They have amazing natural immunity and antibacterial peptides are used as the main component of their host defense mechanism.Studies have shown that insects contain a large number of antimicrobial peptides.At present,305 different types of antimicrobial peptides,which exhibit diversity in their structure and activity,have been isolated and purified from Hemiptera,Coleoptera,Lepidoptera,Orthoptera,Diptera,Isoptera,Homoptera and Acarina.Coridius chinensis belongs to the Hemiptera(Dinidoridae)is an important resource insect.Studies have been proved that the hemolymph of C.chinensis has a strong activity against bacterial,which indicates that the hemolymph contains antimicrobial peptides in its body.At present,antibacterial peptides are fewly reported in C.chinensis.Therefore,we cloned a defensin gene Cc Def from C.chinensis based on full-length transcriptome data in this article.The expression profile and bioinformatics characteristics of defensin gene Cc Def were analyzed,and the antibacterial activity of the mature protein was detected,in order to lay a foundation for the development of antimicrobial peptide resources of C.chinensis and new antibacterial drugs.The main research results obtained are as follows:1.Cloning and characterization of the defensin gene Cc DefThe defensin gene Cc Def was cloned from C.chinensis by reverse transcription polymerase chain reaction(RT-PCR).The c DNA was 432 bp in length and contained an open reading frame(ORF)of 309 bp which consists of 17 amino acid residues of signal peptide,42 amino acid residues of propeptide,and 43 amino acid residues of mature peptide.Moreover,the 5'-non-coding region(UTR)of the Cc Def gene contains 37 bp and 3'-UTR is 86 bp in length.Bioinformatics analysis showed that the Cc Def protein contains two potential phosphorylation sites(T₈₅,T₆₉)and no glycosylation sites.The molecular weight of this peptide is 4656.46 Da,with a theoretical isoelectric point of 7.83 and a total net charge of+1,which participates in the immune protection process of organisms.Mature Cc Def protein contains two negatively charged amino acid residues(D,E),three positively charged amino acid residues(R,K),and six cysteine(Cys)residues,and these Cysteine residues forms three disulfide bonds at positions ⁶²Cys₁-⁹³Cys₄,⁷⁹Cys₂-⁹⁸Cys₅and⁸³Cys₃-¹⁰⁰Cys₆.The mature Cc Def forms a three-dimensional structure containing one?-helix,two?-sheets.Homology comparison and cluster analysis showed that Cc Def was closely related to the defensin of Palomena prasina and Halyomorpha halys.2.Expression profile of Cc DefThe spatio-temporal expression profile analysis of Cc Def gene showed that Cc Def was expressed throughout the developmental stage of C.chinensis.The second nymph was the highest expression,followed by the adult,and the lowest in the 1^st instar nymph.Cc Def was only expressed in fat body and hemolymph,but not in head,integument,muscle,testis and ovary.Moreover,the m RNA expression level of Cc Def was significantly up-regulated and peaked within36 hours after induction by Escherichia coli and Staphylocous aureus.3.Detection of antibacterial activity of Cc DefNucleotide sequence of Cc Def protein was optimized based on the preferential codon usage of Pichia pastoris,which contains the restriction enzyme cleavage sites Xho I,Xba I and yeast Kex2 protease cleavage sites.The gene was cloned into the expression vector p PICZ?A plasmid by T4 DNA ligase and the recombinant vector p PICZ?A-Cc Def expressed in host strain P.pastoris GS115.The results showed that defensin Cc Def induced by recombinant yeast had activity against Escherichia coli.