A Preliminary Study On The Function Of BmVps4 And BmVta1 In Bombyx Mori

Endosomal sorting complex required for transport(ESCRT) machinery is evolutionarily conserved and widely distributed in eukaryotic organism. ESCRT is involved in various processes including cytokinesis, virus budding, autophagy, plasma membrane repair, nuclear membrane reformation, signal transduction regulation, and is also closely related to cancer and neurodegenerative diseases.The ATPase Vps4 is one of key members of ESCRT machinery, and accompanied with its positive regulator Vta1 it can disassemble ESCRTs after execution of their functions for repeated uses. Current research is mainly carried out in yeast and mammalian cells, and much less is explored in silkworm and other insects. In this thesis, we aim to perform a preliminary analysis of the silkworm homologous genes of Vps4 and Vta1, as well as their encoded proteins. The major results are as follows:Using bioinformatic methods, we analyze the silkworm genome and identify the coding genes of Bm Vps4 and Bm Vta1(SGD accession numbers are:BGIBMGA006243, BGIBMGA004694). Bm Vps4 encodes 438 amino acids with a molecular weight of 49 k Da. Bm Vta1 encodes 311 amino acids with a molecular weight of 34 k Da. They are located in the silkworm No.6 and No.22 chromosome respectively. Bm Vps4 contains the N-terminal conversed MIT domain, the middle AAA domain, the small β domain and C-terminal helix. Bm Vta1 contains the conversed MIT domain and VSL domain. Bm Vps4 and Bm Vta1 are highly conserved in evolution through sequence alignment and phylogenetic tree analysis.In order to study the function of Bm Vps4 and Bm Vta1, we have cloned their encoding genes by PCR, constructed expression vector p ET-30a(+)-Bm Vps4 and p ET-30a(+)-Bm Vta1 and successfully expressed the recombinant proteins Bm Vps4 and Bm Vta1 in E.coli BL21(DE3) strain.Using recombinant protein as antigen to immune mice, we have successfully prepared polyclonal antiserum, which can specifically detect the corresponding recombinant protein expressed in E.coli and the endogenous protein of Bombyx mori.The polyclonal antiserum can be used to examine the expression of corresponding protein in silkworm at different development stages and various tissues and organs.Using the report gene technology with expression of EGFP fusions, we have constructed recombinant expression plasmid p IB-Bm Vps4-egfp and p IB-egfp-Bm Vta1 based on insect expression vector p IB/V5-His. After transfecting silkworm Bm N cells with these recombinant plasmids, the expression of these two recombinant proteins has been observed by fluorescence microscopy and they are mainly localized in the cytoplasm.Quantitative Real-time PCR(q RT-PCR), RT-PCR and Western blot have been used to analyze the gene transcription and protein expression levels of Bm Vta1 and Bm Vps4 during silkworm different developmental stages and in different tissues and organs. q RT-PCR and RT-PCR showed that both genes are expressed during the whole life cycle of silkworm, and display highest expression levels during embryonic development and metamorphosis stages. Bm Vps4 is expressed most highly in oosperm with a level of 4 times that in the first instar larvae. Bm Vta1 is expressed highest in moth with a level of 2 times that in the first instar larvae. Consistently, Western blot showed that both proteins are expressed during the whole life cycle of silkworm, and they are significantly up-regulated during embryonic development and metamorphosis stages. Therefore, they may play an important role in the silkworm development.q RT-PCR and RT-PCR also showed that these two genes have certain specific expression patterns in silkworm tissue and organs. They are widely expressed in Malpighian tube, ovary, testis, nerve, head, fat body, midgut, silk glands and hemolymph, but are highly expressed in the head and hemolymph. Western blot analyses further confirmed that these two proteins are expressed widely, but they show highest expression levels in ovary and testis, and their expression levels in head are much lower and cannot be detected even in hemolymph. We are currently trying to solve this discrepancy by optimizing experimental protocols.RNAi has been used to explore the role of Bm Vps4 in the silkworm metamorphosis. The synthetic si RNA fragments was injected into the fifth instar silkworm larvae(fifth day) and could significantly reduce the m RNA level of Bm Vps4,and prominently retarded the metamorphosis process. On the third day after injection,the expression of Bm Vps4 gene was decreased by 61% and 54% compared with the non-injection silkworm group and negative control group(si-egfp), respectively. The body weight growth rate was only 56 % that of non-injection group and 60% that of negative control group. The pupation time was extended to 50 minutes longer than the non-injection group and 40 minutes longer than negative control group. After 13 days of RNAi treatment, compared to control group, the silkworm pupa body weight was decreased, the body length was shortened, the cocoon is smaller in volume with thinner silk fiber. Amazingly, the proportion of silkworms with shortened wings was significantly increased to 30%, which is 3 times that of non-injection group and 2times that of negative control group. Together, these results indicate that Bm Vps4 does play an important role in the silkworm metamorphosis.QRT-PCR has been used to analyze the gene transcription levels of Bm Vta1 and Bm Vps4 during the process of Bm NPV infecting Bm N cells. q RT-PCR showed that the transcription of Bm Vps4 is significantly up-regulated at 50, 60 and 90 min post-infection, and that at 50 and 60 min is higher than at 90 min. Similarly, the transcription of Bm Vta1 is up-regulated at 20, 60 and 90 min post-infection, but that at90 min is higher than that at 20 or 60 min. The results indicate that although the functional roles of Bm Vps4 and Bm Vta1 are closely related, there are still some differences between them.