Basic Study On Fibroin Heavy Chain Gene Of Silkworm, Bombyx Mori And It's Application

We have constructed expression cassettes of EGFP fusion proteins driven bysilkworm fibroin heavy chain (FibH) promoter with different lengths of FibHN-terminal sequence, C-terminal sequence, and introduce these genes into silkwormusing recombinant AcMNPV as transfer vectors, effect of these fragments on thesecretion of the recombinant proteins was studied systematically. Results showed thatthe two α-helices from FibH N-terminal and the 64 amino acid residues fromC-terminal are not necessary for cleavage of signal peptide and secretion of therecombinant fusion proteins in posterior silk gland cells. Fusion protein withN-terminal 163 amino acid residues of FibH, EGFP and a His-tag at its C-terminalwas expressed in posterior silk gland and purified. N-terminal sequencing of thepurified protein showed that the signal cleavage site is between position 21 and 22amino acid residues. Further study on FibH signal peptide by systematicallyshortening from C-terminal showed that with 20, 18, 16, 12 amino acids in length candirect secretion of the reporter, yet with 11, 10, 9, 8 and 1 amino acids in length cannot. When FibH signal peptide was shortened to 12 amino acid residues, the secretionefficiency is decreased slightly and the cleavage happened within EGFP. When 16amino acid residues of FibH signal peptide was used, the secretion of fusion protein isnormal and the cleavage site is between the Gly-Ser linker and the start amino acidMet of EGFP. It is applicable for expression of foreign proteins in silkworm silk glandwith this structure to control the N-terminal amino acid of the mature protein. Thesignal peptide cleavage sites of the recombinant fusion proteins was also discussedand compared with the prediction results of SignalP 3.0.Synthetic silk genes with functional regions from spider dragline and silkwormfibroin heavy chain driven by FibH promoter were successfully expressed in posteriorsilk gland of silkworm using recombinant AcMNPV as gene transfer vector. Theexpressed silk proteins can be secreted normally into silk gland and react specificallywith the spider silk antibody. This may facilitate the further study of improving themechanical property of silkworm silk using biotechnology method.An insertion sequence name BmTH3 was found at the 3'-flanking region of FibHgene. Occurrence of this insertion is different among geographical strains of silkworm.This insertion happens in most Japanese strains and some European strains, but israrely found in Chinese strains and tropical strains. This insertion does not happen inwild silkworm, either. BmTH3 exists at other sites of genome with a high copynumber of all the silkworm strains and wild silkworm investigated. This provides amolecular marker for the study of silkworm origin and silkworm transmission.BmTH3 has a transposon like structure for having a 17 bp ITR (inversed terminalrepeat) and a 5 bp direct repeat at the target site. BmTH3 was proposed to be atruncated non-LTR transposable element as analyzed through the silkworm genomedatabase and EST database.