Molecular Cloning And Expression Analysis Of Ha-hdd23Gene From The Cotton Bollworm,Helicoverpa Armigera

Hdd23gene was first discovered and named in the fall webworm, Hyphantria cunea. After injection of Escherichia coli and Saccharomyces cerevisiae, expression of Hdd23gene was significantly up-regulated. The specific function of Hdd23gene in innate immunity was not clearly by now. In this study, we cloned this gene from cotton bollworm, and named Ha-Hdd23, Using RNA interference technology, we preliminary study about the function in innate immunity of cotton bollworm. The results as follows:1. cloning and sequence analysis of Ha-Hdd23form Helicoverpa armigeraBy using RT-PCR techniques, we cloned a gene from Helicoverpa armigera. Sequence analysis showed that the full length cDNA was544bp, and encodes119amino acids, Theoretical molecular weight of predicted protein is12.9kDa, isoelectric point is6.18, The protein also has a signal peptide from first to22amino acids. Sequence alignment shows that the highest amino acid similarity of39%with Hyphantria cunea. So we named it Ha-Hdd23.2. Tissue specific expression analysis of Ha-Hdd23Extract total RNA of four organizations of Helicoverpa armigera, and use RT-PCR to analysis its expression in this four different tissues:fat body, epidermis, hemolyph and midgut, the result shows Ha-Hdd23gene are expressed in midgut and hemolyph, not expressed in fat body and epidermis.3. The effects on relative expression of Ha-Hdd23after injection of different microbialAfter Injection E.coli and S.auras into the cotton bollworm, The relative expression level of Ha-Hdd23was detected by real time PCR in hemocytes.The result shows that the expression of Ha-Hdd23in hemocytes has no significant changes after inject E.coli and S.auras. These results indicate that Ha-Hdd23may be involved in the regulation of the innate immune.4. RNAi analysisIn order to further study the function of Ha-Hdd23in innate immunity, we used RNAi to interfere the expression of Ha-Hdd23gene. We firstly synthesized the dsRNA of Ha-Hdd23in vitro, then injected it into larvae through micro injection, The results showed that:After injecting Ha-Hdd23dsRNA into1st days4th instar larvae of H. armigera, the expression of Ha-Hdd23gene is significantly lower than the control group in hemocytes after an immune challenge by E. coli bacteria to stimulate the6h and12h, phenoloxidase activity is decreased significantly, a significant reduction in the number of nodules formed. In summary, the experimental from the cotton bollworm blood cells cloned with congenital immune-related gene Ha-Hdd23, based on the experimental results, we hypothesized that the Ha-Hdd23may play an important role in the insect innate immune response, and may be involved in phenoloxidase reaction and nodule formation of blood cells.