Gene Cloning And Expression Analysis Of BmHP14in The Silkworm, Bombyx Mori

Similar to other invertebrates, insects lack an adaptive immune system in higher vertebrates, and solely rely on innate immune systems to recognize injuries and fight against invading microorganisms. The immune systems contain cellular and humoral responses. Cellular immune responses include phagocytosis, encapsulation and nodulation, which are mediated by hemocytes. Humoral immune responses comprise of clotting, melanization, and synthesis of antimicrobial proteins.In the prophenoloxidase (proPO) cascade, a serial of serine proteinases are activated sequentially and the initiate signals are amplified. Activation of the cascade leads to the proteolysis of proPO to produce the active phenoloxidase (PO). The active enzyme catalyzes two kinds of reactions:the oxygenation of monophenols to o-diphenols and oxidation of o-diphenols to the corresponding quinones necessary for the generation of melanin.With the knowledges of prophenoloxidase (proPO) cascade in Manduca sexta, we know that as an initiate immune responses serine protease, MsHP14is auto-activated and catalyzes the downstream proteases. The studies of proPO cascade in Bombyx mori have been carried out earlier, however there are no reports about the initiate protease of proPO cascade in silkworm. In the present study, based on the information of MsHP14and silkworm genome sequences, the BmHP14was cloned and the expressions of this gene were analyzed. The results are as follows:1. The cDNA cloning and sequence analysis of BmHP14Based on bioinformatic analysis, a homology of MsHP14(Manduca sexta hemolymph protein14) was obtained in EST database of Bombyx mori. Its cDNA was cloned by RACE. BmHP14gene has17exons intervened by16introns. The predicted BmHP14protein is71kDa, and its theory isoelectric point is5.09according to the online prediction. The first seventeen amino acids at the N terminal are predicted to constitute the signal peptide using the SignalP3.0Server. The upstream of5’UTR contains regulation sites, such as transcription factor NF-κB binding site and GATA. The clustalX analysis of serine protease genes of Bombyx mori, Manduca sexta, Tenebrio molitor. Drosophila melanogaster shows that BmHP14has a highest conservation with MsHP14. Protein HP14in M.sexta is known to be the first serine protease autoactivated in the proPO activation cascade, and play an important role in the following pathway. We presume that BmHP14is the initial serine protease autoactivated in the silkworm proPO activation cascade.2. Expression analysis of BmHP14The cDNA of3rd day of5th instar larva tissues in silkworm larvae were obtained. RT-PCR analysis indicated that BmHP14is expressed in fatbody, malpighian tube, testis, ovary, cuticle, haemocyte, and head of silkworm. And BmHP14is mainly expressed in fatbody. The expression of BmHP14is up regulated in the silkworm fatbody after the infection of pathogens including Bacillus bombyseptieus, Beauveria bassiana and Escherichia coli, which were investigated by RT-PCR and qPCR.3. Production of BmHP14antibody and Western blottingA recombination protein pET28a-His-BmHP14was produced and served as antigen for the production of polyclonal rabbit antiserum. The adult male rabbit was immuned by purified recombinant proteins. And the antiserum of BmHP14was collected. The Western blotting analysis indicated that the expression of BmHP14protein was up-regulated after pathogen infection including B. bombyseptieus, B. bassiana and E. coli in plasma of Bombyx mori. The results showed that BmHP14protein was up-regulated3hours after infected by B. bombyseptieus, later down-regulated in6hours, then up-regulated until24hours. After infected by E. coli, BmHP14protein was up-regulated in1hour, and maintained a high expression level all the time from1hour to24hours. After infected by B. bassiana, BmHP14protein was up-regulated in1hour to3hours, thereafter the expression level was down-regulated until to24hours. The BmHP14in plasma was up-regulated1hour to3hours after infected by pathogen, indicating that the transcription and expression of BmHP14were quickly regulated in the fatbodies from the infected silkworm larvae. Then the BmHP14proteins were released to plasma and involved in melanize process to defense the pathogens.