Functional Analysis Of Bmcp8, A Small Cationic Protein In Silkworm, Bombyx Mori

Insulin and insulin-like growth factors (IGFs) signal system control growth and metabolism in both vertebrates and invertebrates. As an important effective factor, insulin-like growth factor binding proteins (IGFBPs) play the essential role on the life-process, such as maintaining the balance of blood glucose level, promoting cell proliferation et al. Although researchers found the insulin-like peptide in some insect species, there are still no reports about the functional homologue of IGFBPs in invertebrate.As an important part of the innate immune system, PO (Phenoloxidase) cascade system protects the host from the infection of exogenous microorganisms. This immune response will kill the pathogen through melanin or Quinones, reactive oxygen species, NO, which were the by-products produced during the melanin formation. However, the excessive.melanization would be harmful to the host too. So the negative-regulator in the PO system will need to be required to control the over melanization.Silkworm larvae own the open blood vascular system. Hemolymph protein will be involved in metabolism, mass-energy conversion, immune response, et al. During the functional analysis of BmCP8, the results could be fall into two different divisions:the insulin-like peptide signal and PO cascade system. The main results are as follows:1. Bioinformatics analysis of BmCP8Based on the B1ASTP in the UniprotKB database, the alignment of amino acid sequences of the BmCP8 with A. mylitta fungal proteinase inhibitor AmFPI-1 and N-terminal domain sequence of mammalian IGFBPs founds the sequence similarity, all of them were rich in the cysteine residues. Multiple sequence comparison demonstrated that BmCP8 also contained the reactive site of AmFPI-1 and the sequence "GCGCCXXV" from BmCP8 owns a high similarity with the "GCGCCXXCA" in the conserved region of N-terminal domain sequence of IGFBPs.2. Purification of BmCP8 protein In order to obtain more native BmCP8, we improved the original purification strategy into ammonium sulfate precipitation, twice gel filtration and cation ion-exchange chromatography, which could effectively get more products.3. Phenotype and BmCP8 gene expression pattern analysis after the high-glucose diet treated.From the third and fifth instar respectively, Silkworm larvae were fed on the artificial diet containing 5% and 20% glucose. Investigating gene expression of BmCP8 and Bombyxin A2 was conducted at the fifth instar day 3, following phenotype analysis. The result demonstrated that the high-glucose diet treated silkworm larvae was characterized by the small size and delayed growth. Compared to the control, BmCP8 gene and Bombyxin A2 gene were down-regulated with glucose-treatment.4. Effects of BmCP8 protein on the growth of silkworm ovarian cellsThe BmCP8 protein with different concentrations (0,0.5,1,5,10 ug/hole) were incubated with the silkworm ovarian cells in logarithmic growth phase. In this experiment, the silkworm cuticle protein CPR56 was taken as the protein control. After being incubated for 48 h, the growth of the ovarian cells was measured. The results showed that BmCP8 can not stimulate the growth of ovarian cells.5. Effects of BmCP8 on the PO cascade system.Injecting fifth day 3 silkworm larva with E. coli, B.bassiana and B. bomyseptieus, the gene expression of BmCP8 was analyzed by q-PCR. Results revealed that the expression of BmCP8 was suffered an acute up-regulation after infected with B. bombyseptieus one hour later. The expression of BmCP8 will be gradually reduced during the following hours. But compared with the control, the expression of BmCP8 still stayed in a high level even at the later 24 hours. The experiment data demonstrated that BmCP8 will quickly and persistently response to the infection of B. bomyseptieus. Meanwhile, the experiment proved that BmCP8 own the ability to inhibit the PO system. When 2ug/ul BmCP8 were add to the SLP reagent which were induced by PGN and BGN, the effect of melanism will be repressed by BmCP8.