Cloning And Expression Of BHLH, The Transcription Factors Expressed Specially In The Silkgland Of Silkworm, Bombyx Mori

Fifth instar larvae of Bombyx mori can spin a large number of silk protein.Silk protein is formed of fibroin protein and sericin protein by a certain ratio.Fibroin protein is producted at PSG and sericin protein is producted at MSG.At molting and metamorphosis stage,the expression of silk genes at a low level.Scientists found a large number of transcription factors in silkgland,these factors paly roles in the silkgland development,silk gene transcription and cell apoptosis etc. Most silk gene transcription factors belong to HOX and FOX family,the bHLH transcription factors are uncommon.As the data of Bombyx mori genome and gene chips published,we got a research platform to study silkgland transcription factors.Based on gene chip data retrieval,two factors—Bmdimm and Bmsage which have bHLH domain were found,bHLH factors have a helix-loop-helix domain,they can control target gene's expression by combined with DNA specifically.Bmdimm express in PSG of silkgland,Bmsage express both in MSG and PSG.We conject they may involved in the regulation of silk gene.In order to research the function of Bmdimm and Bmsage,we clone and analysise Bmdimm and Bmsage,express these protein in vitro and determine the site of gene expression by In situ hybridization.The main research contents and results are as follow:1.Bioinformatic analysis of Bmdimm and BmsageBmdimm and Bmsage are 3649bp and 2435bp in length,both of them have 4 exon and 3 intron. Bmdimm is on Chromosome 17.Bmsage is on Chromosome 25 which fib-H is located on. We predict the protein domain of Bmdimm and Bmsage in SMART,and conform that Bmdimm and Bmsage gene belong to bHLH family transcription factor.2.Expression feature of Bmdimm and Bmsage and tissue in situ hybridizationBased on the silkworm microarray database and mRNA level analysis,the expression profile of Bmdimm and Bmsage(Dazao,day three of fifth instar) in different tissue is as follows:Bmdimm expressed in PSG while Bmsage in MSG and PSG,and they have low or nearly no expression in other tissues.From 4^th to 5^th,the expression of Bmdimm and Bmsage showed an in creasing tendency.As the silk gene no longer expressed,the expression of Bmdimm and Bmsage was downtrend.We also found that Bmdimm not only express in PSG but also in MSG in molting stage of 4^th.In embryonic period,Bmsage expresses with a rising trend as embryonic develop,but Bmdimm has no expression.We locate Bmdimm and Bmsage on silkgland by in situ hybridization.The experiment shows that,we get the signal of Bmdimm and Bmsage in PSG while Bmsage in MSG.The result was consisten of the Bmdimm and Bmsage expression features on mRNA level.3.Cloning and prokaryotic expression of Bmdimm and BmsageWe designes primers and cloned the coding sequence of Bmdimm and Bmsage into the pET-50b(+) expression vector,and the recombinant plasmid was transformed into Escherichia coli BL21.Then induced recombinant plasmids to express protein by IPTG,we got a soluble recombinant protein at the codition of 30℃,1 mmol/L IPTG induced 3 hours through optimization. At last we purified the protein by affinity chromatography.