Molecular Cloning and Expression Pattern Analysis of a Protein Disulfide Isomerases (SpLPDI) in Spodoptera litura

【Objective】The objective of this study is to analyze the mRNA expression characteristics and immune related function of protein disulfide isomerase gene(SpLPDI) from Spodoptera litura,and to provide a foundation for further study of S.litura immune molecular mechanism in response to Nomuraea rileyi infection.【Method】The full-length cDNA of SpLPDI was amplified by specific primers based on the EST from the fatbody suppression subtractive hybridization(SSH) library and SMART RACE cloning technology and further analysed by bioinformatics software.The expression patterns of SpLPDI in different tissues were analyzed by semi-quantitative RT-PCR and quantitative PCR.The mRNA expression of SpLPDI upon N.rileyi challenge was studied by qPCR and the protein characteristics by prokaryotic expression was analyzed.【Result】The full length cDNA of SpLPDI was cloned which was 2 367 bp,including 1 485 bp ORF encoding 494 amino acid proteins.The SpLPDI protein had two thioredoxin domains and an ER retention signal(KDEL) with a signal peptide of 17 amino acid residues containing an a-b-b′-a′domain organization.The result of multiple alignment indicated that a,a′ domains and ER retention signal were highly conserved.Phylogeny analysis showed that SpLPDI had a closest genetic relationship with Bombyx mori while a distant genetic relationship with Cryptosporidium parvum and Aspergillus niger.Expression profile analysis showed that SpLPDI was expressed in all detected tissues,but highest expressed in hemocytes,followed in fatbodies,and lowest in heads.Induction expression analysis showed that mRNA levels of SpLPDI were significantly increased in fat bodies and midguts upon N.rileyi challenge but the rate of increase in fat bodies higher than that in midgut.SpLPDI was successfully expressed in E.coli,producing a soluble fusion protein.【Conclusion】 The full length cDNA of SpLPDI was cloned from S.litura and changes of PDI mRNA expression were observed in different tissues of S.litura after N.rileyi injection.It suggested that SpLPDI might be related to immune response of S.litura to N.rileyi.