Analysis Of Sericin-1 Gene Promoter Of Bombyx Mori

The Bombyx mori (silkworm) is a silk-spinning domestic lepidopteran insect with both economic and scientific importance. Silkworm has been wildly researched as the model organism of lepidopteran insects because of its relative better research background and convenient culture conditions among the insects. The silk glands are functionally divided into three distinct compartments, the anterior (ASG), middle (MSG) and posterior (PSG) silk glands. Fibroins, the fibroin L and H chains and fibrohexamerin (formerly known as P25), are expressed exclusively in posterior silk gland (PSG), whereas the sericins are produced specifically in MSG. In this regard, the silk gland of Bombyx mori constitutes an attractive model for studying mechanism governing spatially and temporally programmed transcription.There are five kinds of sericin genes are cloned and separated, there names are Ser1, Ser2, MSGS3, MSGS4 and MSGS5. Sericin is a group of glue proteins which ensure the cohesion of the cocoon by sticking the silk threads together. Six kinds of sericin protein molecules are coded by two genes, ser1 and ser2, through alternative splicing. The Ser1 gene is the major constituents of sericins. Recently reports have shown that there are some silkworm strains susceptible to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), which could serve as a novel in vivo gene delivery tool for the silkworm. Therefore developments in recombinant DNA technology and the advent of in vivo baculovirus expression system offer an approach to the study of the molecular mechanisms of transcriptional controls in silk gene in vivo.Owning to the poor understanding of mechanism responsible for the tissue-specific expression of the Ser1 gene, Liu Y.'s studies were carried to analyze the related region in the sericin-1 gene promoter, and address to explore the possible cis-acting region(s) that takes part in determining the tissue-specific expression of the sericin-1 gene in vivo.To study Ser1 gene exactly, we constructed a dual-luciferase reporter donor plasmid, which have both firefly (Photinus pyralis) luciferase and Renilla (Renilla reniformis) luciferase. Renilla luciferase (RLuc) was driven by Bombyx mori actin3 promoter, firefly Luciferase (FLuc) was driven by the promoter which we want to studies. We amplified 5 kinds of Ser1 promoter which truncated in 5'terminal by polymerase chain reaction (PCR), torether with the full length (1.6 kbp) of Ser1 promoter, 6 Ser1 promoters with different lengths were cloned into dual-luciferase reporter donor plasmid. They were introduced into the Bacmid AcNPVΔEGT to generate recombinant AcMNPV. Studies showed that -1600~-475 haven't factor is necessary for sericin-1 promoter.To analyze contribution of several regions -475 bp upstream sequence, 3 kinds of recombinant AcMNPV with different mutantions in -475 Ser1 promoter were constructed. The Ser1 promoter was studied in vivo using those recombinant AcMNPVs, results showed that SA fragment and CAAT box aer necessary for sericin-1 promoter.In general, the present studies may not only contribute to understanding the mechanisms of Ser1 gene expression and benefit to improving the future production of silk-manufacture industry, but also do good to develop silk gland bioreactor in the future.