Role Of Ha-SRP2 Gene In Innate Immunity Of The Cotton Bollworm,Helicoverpa Armigera

The relevant reports show a SRP gene which named Hdd23 before was identified as one of genes up-regulated in the fall webworm, Hyphantria cunea.Then, we identified and characterized a gene that had high similarityto Hdd23 genefrom the common cutworm, Spodoptera litura. The study found that enhanced gene expression was observed in larvae under severe stress conditions:abdominal ligature, proleg cutting, mechanical vibration, low temperature, and heat shock. So, we named it stress-responsive peptide, SRP. Recently, we identified a SRP gene (Ha-SRP) by sequencing the transcriptome of the cotton bollworm, Helicoverpa armigera, hemocytes. Through the relevant research, we hypothesized that the Ha-SRP may play an important role in the insect innate immune response, and may be involved in phenoloxidase reaction and nodule formation of hemocytes. Further more, we identified a SRP gene by sequencing the transcriptome of the cotton bollworm, Helicoverpa armigera, hemocytes, and named Ha-SRP2. The SRP gene which was identified before named Ha-SRP1. In order to research the specific function of Ha-SRP2 in the innate immunity, We used prokaryotic expression, protein function analysis, and RNAi. We got the following results:1. Gene cloning and sequence analysis:Gene Ha-SRP2 contains a ORF of 360 bp. And the predicted protein encoded by Ha-SRP2 is 119 aa long.The predicted MW of rHa-SRP2 is 12.799kDa, and the isoelectric point is 6.04. The proteins contain a signal peptide sequence, no transmembrane region sequence, show that the protein is a kind of secretory proteins. A multiple sequences alignment indicates that Ha-SRP2 amino acid have significant similarities with SRPsequences from other lepidopterans.2. Tisse distribution of Ha-SRP2 mRNAs:Expression of Ha-SRP2 mRNAs were examined using RT-PCR and qRT-PCR in five tissues of H. armigera larvae. Ha-SRP2 was expressed in all tested tissues, which was expressed at a higher level in hemocytes and malpighian tube.3. After Injection E.coli and S.auras into the Helicoverpa armigera, the relative expression level of Ha-SRP2 was detected by real time PCR. The result shows that the expression of Ha-SRP2 is significant up-regulate. These results suggest that Ha-SRP2 may be involved in the regulation of the innate immune.4. Prokaryotic expression and purification of recombinant protein:cloning the cDNA of Ha-SRP2 mature peptide,and connecting it to the expression vector pET-32a. We constructed the expression vector of pET-32a-Ha-SRP2 and import it into E.coli, then expressed the recombinant protein in E.coli and purified by Ni column, finally obtained the recombinant protein rHa-SRP2.5. To examine the biological activity of rHa-SRP2, purified recombinant protein was prepared. Subsequent analysis showed that:(1) rHa-SRP2 could bind Gram-negative Escherichia coliand Gram-positive Staphylococcus aureus; (2) rHa-SRP2 could enhance prophenoloxidase activation in the presence of microbes; (3) rHa-SRP2 promoted the formation of melanotic nodules in vivo; (4) rHa-SRP2 could bind hemocytes.6. Moreover, RNA interference assays were performed to further confirm the function of Ha-SRP2 gene. When this gene was knocked down by dsHa-SRP2, the phenoloxidase activity in bacteria-challenged larval hemolymph was significantly decreased and the number of nodules are significantly reduced, leading to the Clearing ability of hemolymph to E.coli were significantly decreased.These results indicated that Ha-SRP2 may play an important role in the insect innate immune response, and may be involved in phenoloxidase reaction and nodule formation of hemocytes.