Cloning And Functional Analysis Of Lectin Genes From Chinese Oak Silkworm,Antheraea Pernyi

Lectins as an important pattern recognition receptor,play an important role in the insect immune system.Lectins can identify specific molecules on the surface of pathogenic microoganisms,thus activating the insect immune defense system.Different lectin contains different specific carbohydrate recognition domain CRD and identify specific molecular molecules on the serface of pathogenic microogainsms.Among them,C-type lectin is a major family of Lepidoptera,it is a calcium-dependent glycoprotein.It has good binding ability to glycoproteins and glycolipids on the surface of pathogens.This research was a study about the immune function and mechanism of lectins from Chinese oak silkworm,Antheraea pernyi.In order to clarify the function and mechanism of lectin,we carried out bioinformatics analysis,tissue expression analysis,expression level change analysis with diffierent microorganism infection,and agglutination activity analysis of recombinant protein.1.Based on the previous transcriptome database established by our laboratory,we cloned four genes encoding C-type lectins from A.pernyi.Bioinformatics methods were applied to analyze the sequence structure and evolutionary relationship.Four lectin genes from Chinese oak silkworm were cloned,named as Lectin,Lectin16,Lectin21,CTL,respectively.Open reading frame of Lectin was 678 bp which encoding 225 amino acids.The isoelectric point(PI)and protein molecular weight was 6.30 and 26.11 k Da,respectively.The signal peptide was 1-19 amino acids and no transmembrane structure.Genes of Lectin16,Lectin21,CTL contained ORF of 708 bp,807 bp and 924 bp coding for protein of 235 bp,245 bp,307 bp,respectively.The predicted protein of Lectin16 and CTL constructed of 34-and 15-residue signal peptide sequence,and there was no transmembrane structure.Both of four had the common characteristics of C-type lectins with the CRD catalytic active domains.2.The semi-quanfitative RT-PCR is used to analyze the profile in different development and tissues.Semi-quantitative PCR analysis indicated that four genes were mainly expressed in fat body,midgut and gonad.Among the four tested lectins genes,CTL was widely distributed in the tissues of 5th larvae,except for plasma and malpighian tubule,the epidermis had the high expression.And others had the high expression in plasma,midgut,fatbody,testis and ovary.3.The Real-time PCR is used to analyze the expressions induced by exogenous stimulus over periods of time.Under infection of five different types of pathogenetic microorganisms(Nosema pernyi,Antheraea pernyi Nuclear Polyhedrosis Virus,Escherichia coli,Beauveria bassiana and Streptococcus pernyi),the expression levels of lectin genes in the midgut tissue were induced significantly.And the expression levels of lectin genes varied more sensitively with the maximum transcripts level at 6-9 h by E.coli and NPV.After infected with Nosema pernyi,the expression enhanced with the increasing infection time and reach the maximum at 24 h.When infected with Streptococcus pernyi,the expression levels of lectins in hemocytes were reaching the maximum value at 9 h and diminished gradually.After 24 h infected with Beauveria bassiana,the lectin genes began to express at a high level.4.The cloned gene was ligated with prokaryotic expression vector p EASY?-E1 Expression vector and transformed into competent cell BL21(DE3).The results of SDS-PAGE analysis showed that the recombinant plasmid induced by IPTG could express the fusion protein which was consistent with the predicted protein molecular weight.The results of western bolt analysis were consistent with the result of SDS-PAGE analysis.The agglutination results showed that the recombination protein had agglutination activity against E.coli and rat red blood cells.