Functional Analysis Of Cis-acting Elements Of Silk Fibroin P25 Protein Gene's Promoter From Silkworm, Bombyx Mori

The expression of silk genes has the apparent specificities of tissue and developmental stage during the whole developmental process of silkworm larvae, of which the interaction of cis-acting elements and trans-acting factors plays an important role on the expression and regulation of silk genes. Study of the gene encoding P25 protein, particularly the cis-acting elements of promoter region, plays a crucial role in further analysis of the spatial- and temporal-specific expression of P25 gene and thus promotes the research of silk genes'expression and regulation.In this paper, firstly, we analyzed the expression of three fibroin genes in different tissues and at different developmental stages using quantitative real-time PCR method. From the results we found that the expression of three fibroin genes was very high in posterior silk gland during the fifth instar. Except for posterior silk gland, there was a certain degree of the expression of fibroin genes in other tissues, such as fat body and middle silk gland; at the fourth molting stage there also existed the expression of fibroin genes, of which the expression of Fib-L was much higher than that of the other during this stage. At the same time, we found that the ratios of three fibroin genes'expression in PSG3 and PSG5 were 9:18:1 and 19:32:1 respectively at the level of mRNA; there existed a coordinated relationship between three fibroin genes'expression at the level of mRNA, especially between Fib-H and Fib-L. Through the quantitative analysis of three fibroin genes'expression in different tissues and at different developmental stages of silkworm larvae, especially the quantitative analysis of P25 gene's expression, it provided a statistical basis for the next functional analysis of cis-acting elements of P25 gene'promoter.Secondly, we analyzed the activity of upstream 1233 bp of P25 gene's promoter and its regulatory elements'function by using of Bac-to-Bac expression system and fluorescent real-time quantitative PCR technique and so on. The results showed that there might have positive regulatory elements in P25 promoter's upstream of -423 to -1233 and -127 to -238, and in the region of -423 to -238 there might have negative regulatory elements. The two regulatory elements of PSGF and BMFA in P25 gene's expression might play a negative regulation role. The regulatory element of PSGF had a certain enhancement to the activity of A3 promoter in posterior silk gland and it further validated the function of PSGF regulatory element. Analysis of P25 gene promoter's activity, in particular the functional analysis of PSGF and BMFA regulatory elements, could be in favor of further understanding the mechanism of P25 gene's expression and regulation. Finally, we expressed P25 protein in insect Sf21 cultured cells and silkworm by using the AcMNPV Bac-to-Bac recombinant baculovirus expression system, and made a preliminary exploration for the conditions of P25 protein's expression and purification. As results showed that, P25 protein with signal peptide and without signal peptide could all be expressed normally, and the former could be secreted into cell culture medium or hemolymph of silkworm, while the latter mostly stayed in the cultured and silkworm cells. We purified fibroin P25 protein with a His tag from the body of silkworm by using Ni resin. The initially purified conditions for exploration were that: the concentration of imidazole was 100 mM in Wash Buffer, and was 500 mM in Elution Buffer. Through the analysis of fibroin P25 protein's expression in the cultured cells and silkworm, as well as the exploration of conditions of expression and purification, they can make for the further study of P25 protein's structure and function.