Characterization Of The Insulin-like Peptides Gene In Bombyx Mori(BmILP)

Insulin/Insulin-like growth factors play important roles in promoting proliferation, differentiation, growth and development of organisms. The silkworm, Bombyx mori, is an important model organism. The full genome sequencing work of the silkworm had been completed in 2003. So it is convenient for us to study the function of insulin-like peptides by using silkworm. In invertebrates, the first insulinlike peptides was identified in the silkworm, and was named bombyxin eventually. Then many of the insulin-like peptides were identified in other invertebrates, such as flies, mosquitoes, butterflies and bees.So far there were a total of 38 bombyxin genes and one Bombyx IGF-like peptides(BIGFLP), which exist in the silkworm genome. These genes have twelve families, of which seven families A-G and five families V-W, respectively. The family contains 10 members of bombyxin A, 12 members of bombyxin B, 6 members of bomyxin C, 2 members of bombyxin V and only one member for each of bombyxin D, E, F, G, W, X, Y, and Z. Studies of bombyxin were mainly focused on gene level, but little about protein level and physiological functions in the body of the silkworm. The main results and contents of this research are as follows:(1) Bioinformatics analysis showed the gene lengths of BmILP were 4127 bp, which appeared to be on chromosome 1 of the silkworm genome, and it included 5′-UTR, 3′-UTR, two introns and three exons. Its ORF contained 339 bp and coded for a protein having 112 amino acid residues. The predicted molecular weight of Bm ILP is about 12.6 kDa. In the front of Bm ILP was a signal peptide from 1 to 15 amino acids and the signal peptide sequence of 5′ end domain of the mRNA can formed a complex secondary structure. Amino acid similarity analysis of BmILP and bombyxin families showed that BmILP and bombyxin Z1, as well as bombyxin B3 and B12 were the same peptides. Conservation analysis showed that Bm ILP was similar to the members of the bombyxin family, which all contain cysteines at the same positions and are highly conservative. It also indicated that the cysteines were very important to produce higher structure of BmILP and bombyxin families. Motif analysis showed that Bm ILP contains two motifs, which demonstrated that Bm ILP was highly conserved during evolution as the same as bomyxin families.(2) The ORF specific primers were used to amplify the fragment of BmILP gene. The full-length BmILP gene and the gene without the signal peptide were cloned and expressed using expression plasmid pET-30a(+) in E.coli BL21. It was noted that the signal peptide had significant influence on the expression level of the BmILP gene and significantly decreased the expresion level, which may be caused by the formation of the complex secondary structure on the 5′ end of the mRNA, thereby hindered the initiation of the translation. Then the fusion protein was purified and dialyzed for renaturation, and the purified protein was identified by MALDI-TOF mass spectrometry. After the concentration of the purified fusion protein was detected, the pufified protein was used to immune healthy mice and prepare Bm ILP polyclonal antibodies.(3) Quantitative real-time PCR showed that the BmILP gene was produced in diverse tissues of the fifth instar larvae and all developmental stages of the silkworm. BmILP was predominantly expressed by the ovary among different tissues in the silkworm, followed by the fat body, whereas its expression level was very low in the head, silk gland, midgut, testis and malpighian tubule. Particularly, the adult females were found to have higher expression levels than the adult males, and the expression level appeared to be the highest in the mated female moths, suggesting that BmILP may be a key factor in the regulation of egg maturation in Bombyx mori.(4) The His-tag pull down experiments indicated that there might be an interaction between BmILP and the autophagy-related protein Atg8, indicating that BmILP may play a role in immunity. In addition, the immunoprecipitation test was conducted with BmILP polyclonal antibodies to find that BmILP may be formed multimer in eukaryotic cells. But further experimental verification is needed.(5) After the BmILP gene was interfered by siRNA, the results of RNA interference was detected by quantitative real-time PCR and found it is successful. The results showed that the transcription level of BmILP gene begin to decrease from 24 hours and continued until 72 hours, then it went back to normal at 96 hours. When the expression level of BmILP gene began to rise from 24 h to 72 h, conversely, the total sugar content of hemolymph began to decrease and can not affect the variety of weight, which demonstrated that Bm ILP can decrease the toal content of hemolymph in silkworm and it was speculated that BmILP may be involved in the process of glucose metabolism in silkworm.