Identification And Expression Analysis Of QM-like Gene And PDI-like From Spodoptera Litura After Challenge By Nomuraea Rileyi

Spodoptera litura Fabricius is an important worldwide polyphagous agricultural pest and found mainly in the Yellow River basin and the Yangtze River basin of China. S. litura has an extensive host range, covering more than290host plants which belong to99families and most of which are cruciferous vegetables plants.The long-term irrational application of chemical pesticides has caused many problems, such as pesticide residues in vegetables, pests resistance to insecticides and ecological unbalance. Hence biological control is being paid more attention to, especially microbial pesticides. Nomuraea rileyi is one of valuable entomopathogenic fungus and able to cause epizootic diseases in various insects, especially in noctuidae insects.The study on the molecular mechanisms of immune defense in S. litura against N. rileyi has a very important significance in development of new N. rileyi fungicides. In the present study, a QM-like gene and a PDI-like gene were isolated by fat bodies SSH library from N. rileyi challenged Spodoptera litura larvae. We sought to define potential innate immune function of them upon N. rileyi challenge. The results are as follows:(1) About OM from S. litura (SpLQM)①Based on EST of QM, the full-length cDNA was cloned from S. litura using SMART RACE RT-PCR, whose amino acid sequence was identified by ORF finder. The physicochemical properties and protein structure of deduced amino acid were analyzed using SMART and Expasy etc. software. The QMs amino acid sequences similarity percentages of different organisms were calculated using the MegAlign program. And then multiple alignment and phylogenetic tree construction were performed by software Clustalw and MEGA, respectively. The full length cDNA of QM (SpLQM) is838bp, including660bp ORF encoding219amino acid proteins with a molecular weight of25.6kDa and a theoretical isoelectric point of10.0. Analysis of the SpLQM protein sequence was showed that there were presence of several characteristic motifs, including protein kinase C phosphorylation motifs, N-myristoylation sites and amidation motifs in the deduced SpLQM protein, but neither a signal peptide nor nuclear localization signal (NLS) was detected. Homology comparison and phylogeny analysis showed that SpLQM had99.1%and97.7%identity to QMs of Manduca sexta and Mythimna separata, respectively and the genetic distances were the smallest while it shared60.8%similarity with Saccharomyces cerevisiae with the lowest genetic distance, indicating the high level of gene conservation. Results of Clustal W alignment indicated that the ribosomal protein L10signature and SH3-binding motif were highly conserved; the N-terminal and internal regions of SpLQM were more conserved than the C-terminal region. Genomic structure analysis showed SpLQM gene contains three introns.②Tissues specific expression of SpLQM in S. litura was analyzed by RT-PCR and RT-qPCR. Analysis of the tissue expression pattern showed that the SpLQM mRNA was expressed in all tissues tested and the relative expression level of hemocytes was the highest (58times more than that in head), followed by fat bodies (8times more), while the lowest was detected in cuticles.③After incubation with conidial suspension of N. rileyi,the hemolymphs collected from S. litura at24h,48h,72h were observed with the microscope, and normal hemolymphs as control. The results showed most spores were not germinated in hemocoel of S. litura at24h post injection (hpi) and then spores started to reproduce by fission at24hpi. While hyphalbody was rapidly reproducing at72hpi, the hemocytes were significantly declining, most even bursted. At the same time, the expression property of SpLQM was detected by RT-qPCR. SpLQM mRNA expression in fat bodies, hemocytes and midguts of S. litura increased in different degrees. The SpLQM in fat bodies gradually went up from6h to12h and peaked at24h post N. rileyi challenge (4.6folds), followed by a drop at48hpi; while in hemocytes, its transcript began to rise6hpi, then peaked at12h (3.3folds), and afterwards gradually declined from24h to48hpi. The expression level of SpLQM was also up regulated in midguts, but not as high as the levels in hemocytes and fat bodies.④The coding region of SpLQM was cloned again after analyzing the restriction enzyme site, and then subcloned into the pET30a (+) expression vector, resulting in recombinant expression vector pET30a-SpLQM. After sequenced confirmation, pET30a-SpLQM was transformed into E. coli BL21(DE3pLYsS strain). Fusion protein expression was induced by isopropyl-β-d-thiogalactoside (IPTG). Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the recombinant SpLQM expressed in E. coli was predominantly insoluble and then purified by gel extraction kit. Western blotting analysis demonstrated that SpLQM was successfully expressed in E. coli and purified.(2) About PDI from S. litura (SpLPDI) SMART RACE RT-PCR and further analysed by bioinformatics software which is the same as described above. The result showed that the full length cDNA of PDI (SpLPDI) was2367bp, including1408bp ORF encoding494amino acid proteins with a molecular weight of55.3KDa. The SpLPDI protein had two thioredoxin domains and an ER retention signal (KDEL) with a signal peptide of17amino acid residues containing an a-b-b’-a’-c domain organization. The Result of multiple alignment indicated that a, a’domains and ER retention signal were highly conserved. Phylogeny analysis showed that SpLPDI had83.0%identity to PDI from Bombyx mori and the genetic distance was the smallest while the farthest genetic relationship to Cryptosporidium parvum and Aspergillus niger.②Tissues specific expression of SpLQM in S. litura was analyzed by RT-PCR and RT-qPCR. Expression profile analysis showed that SpLPDI was expressed in all detected tissues, highest expressed in hemocytes (110times more than that in head), followed in fatbodies (41times), and the lowest in head, suggesting that SpLPDI might be related to the specific functions of these organizations.③Induction expression analysis showed that mRNA levels of SpLPDI were significantly increased in fat bodies and midguts upon N. rileyi challenge; the relative expression in fat body reached its maximum height at24hpi while the highest level in midgut appeared at12hpi. Besides, the rate of increase in fat bodies higher than that in midgut, indicating that SpLPDI might perform immune-related functions against fungi infection.④The coding region of SpLPDI was cloned again after analyzing the restriction enzyme site and removing the signal sequence, and then subcloned into the pET30a(+) expression vector, resulting in recombinant expression vector pET30a-SpLPDI. After sequenced confirmation, pET30a-SpLPDI was transformed into E.coli BL21(DE3pLYsS strain). The recombinant SpLPDI was obtained after IPTG induction. SDS-PAGE analysis showed the fusion protein SpLPDI expressed in E. coli was predominantly soluble and then purified via Ni-NTA affinity chromatography. The relatively pure fusion protein was collected, whose weight was basically identical to the prediction.Conclusion:Both SpLQM and SpLPDI genes were firstly cloned from S. litura and their tissue expression patterns and induction expression patterns after N. rileyi challenge were then analyzed; both protein characteristics was analyzed by prokaryotic expression. The result indicated that both genes might play an important role in the innate immunity of S. litura in respond to N. rileyi infection.