Studying On Cloning, Expression Characteristics And Function Of Vacuolar-type Atpase In Silkworm, Bombyx Mori)

Silkworm, Bombyx mori, is a kind of important economic insects after artificial domestication, which is considered to be an ideal model organism of Lepidoptera insects in current research. Silk industry is the traditional industry in China with a long history of more than five thousand years, which plays an important role in the social economic and cultural life. At the same time, silk is a kind of biological material with excellent mechanical properties and stable biological inclusiveness, which makes silk products more and more widely used in our life. The studies of silk fiber properties also have been under the spotlight.Silk glands are the sites for synthesis and secretion of silk protein. In the process of fiber formation, silk proteins are synthesized in posterior silk glands and middle silk glands, and are transfered to anterior silk glands(ASG). Along with the change in the metal ions, and proton concentration and the effect of shear forces, silk protein transforms its state from sol to gel in the ASG. Studies carried out in vitro have shown that the secondary structural transition of silk protein is closely related to the change of p H value in different sections of silk glands. Research has proved that Vacuolar-type ATPase(V-ATPases) is associated with the acidification process of silk glands in silkworm. So current research indicates V-ATPases mediate p H reduce in silk gland of silkworm and lead to silk protein fibrosis.V-ATPase is a large multisubunit enzyme with 14 subunits, which consist of two complex domains V1 and V0. V-ATPase participates in a variety of important functions in the cell, such as acidification, cell receptor mediated endocytosis, post-translational translocation, and some specific fusions of organelles and the plasma membrane. Each subunit of the V-ATPases plays an important role for enzyme function. V-ATPases function as proton transportase, transporting protons and affecting the p H indirectly. Recent studies have reported that silkworm V-ATPase has an integral role in silkworm anti-virus process.Although V-ATPase can regulates the p H in the silk glands and influences the formation of silk fiber, the details of this process is still unclear. In addition, the relationships among V-ATPAse, p H and the mechanical properties of silk fiber is still unknown. In order to illustrate the role of silkworm V-ATPase(Bm V-ATPase) in silk fiber formation, this study selected Bm V-ATPase A subunit gene and Bm V-ATPase B subunit gene as the target genes. Firstly, these two genes were cloned and their coded proteins were expressed through the prokaryotic expression system. Then, the polyclonal antibody of Bm V-ATPase B subunit was prepared. Then we analyzed the expression patterns of Bm V-ATPase B subunit in silk gland using this polyclonal antibody. And then, we used RNAi and inhibitor treatment to explore the influence of Bm V-ATPase on cell cycle. Finally, we injected the V-ATPase inhibitor to study the effects of Bm V-ATPase on the mechanical properties of silk fibers. The main results are as follows:1、Gene cloning, expression and purification and polyclonal antibody preparationFirstly, we cloned Bm V-ATPase A and Bm V-ATPase B genes from the midgut on the 3rd day of the 5th instar silkworm larva. Then we conducted TA cloning and obtained recombinant plasmids containing the target gene fragments, and we constructed prokaryotic expression vectors of Bm V-ATPase A and Bm V-ATPase B. After transformed the recombinant vectors to Transetta(DE3) strains, the recombinant proteins and induced by IPTG. We found that the recombinant proteins were expressed in inclusion body under the condition of 37 ℃. After mass spectrum identification of the recombinant protein, we purified Bm V-ATPase B by the method of Ni-NTA affinity chromatography, and used the purified recombinant protein to immunize rabbits. Finally we generated the polyclonal antibody of Bm V-ATPase B with high titer and good specificity.2、Expression patterns of Bm V-ATPase BBecause V-ATPase is a large multisubunit composite enzyme with 14 subunits, the expression patterns of each subunit are the same as the holoenzyme. Therefore, we used the V-ATPase B subunit as a target to analyze the expression patterns of V-ATPase. Firstly, we analyzed the expression patterns of Bm V-ATPase B subunit in different sections of silk gland from the silkworm on the 3rd day of 5th instar and 1st day of wandering stage by western blotting. The results suggested that Bm V-ATPase B subunit was expressed in all sections of the silk gland and it was abundant in the anterior silk gland(ASG) both in these two developmental stages. Furthermore,immunofluorescence indicated that Bm V-ATPase B subunit was located in the silk gland cells layer. Laser confocal scanning microscopy analysis revealed that Bm V-ATPase B subunit was mainly expressed in the cytomembrane of silk gland cells.3、The impact of Bm V-ATPase in cell cycleIn this part, we explored the role of Bm V-ATPase in regulation of the cell cycle. Firstly, we knocked down the gene expressions of Bm V-ATPase A and Bm V-ATPase B in cells by RNAi, and found that RNAi promoted the cell cycle transformation from G1 phase to S phase. On the other hand, the cells were treated by the specific inhibitor of V-ATPase(Bafilomycin A1). And this also promoted the cell cycle transformation from G1 phase to S phase. These results demonstrated that the V-ATPase affected the cell proliferation and division.4、The function of V-ATPase on the formation of the silkWe injected the specific inhibitor of V-ATPase into wandering silkworm larva. Firstly, we observed the morphology of cocoons and measured the diameter of silks. We found that the shape of cocoons became irregular after injecting the inhibitor. Then we detected the diameter of silk reeled from the cocoons by electron microscope, and found that the fibers were thicker. And then we detected the silk fiber secondary structure by FTIR, the results showed that the content of β-sheet reduced after injection, while the content of α helix increased. Finally, we detected the mechanical properties of the silk and we found that the fiber toughness and extension increased, but the strength of silk fiber reduced. These results indicated that V-ATPase can influence the secondary structure and mechanical properties of silk fibers.