Expression And Regulation Of Several Key Gene Related Silk Protein Synthesis Of Silkworm, Bombyx Mori

Silkworm (Bombyx mori) is an insect Lepidoptera Saturniidae moth silkworm, domesticated by the wild silkworm. Bombyx mori feeds on mulberry, spinning silk and breeding widely in China. Sericulture in China has been thousands of years of history. Silkworm is a key economic insect, which made important contributions to cultural development in human society. And silkworm is also a model insect in research. Silkworm has super ability to synthesize silk protein. After less than a month, a silkworm feeds on mulberries about 22g, synthesizes and secretes 0.5～1g of silk protein. Silk protein is made of fibroin and sericin. The expression of silk protein gene is strictly subject to time and space constraints, which sericin protein is largely synthesized in middle silk gland and fibroin protein is highly expressed during the middle and late fifth instar. In the expression and regulation of silk protein gene, it is concerned that the "open" and "close"expression pattern of silk protein gene is a typical research model in eukaryotic transcription and translation.70s and 80s last century, many scientists made a huge number of research on silkworm. Until now, it is seldom known about the ability to be effectively synthesized in silk gland and the precise regulation mechanism which a gene is expressed and synthesized. Silkworm (Bombyx mori) is the first Lepidoptera insect that is whole genome sequencing. This research analyzes and studies the specifically expressed genes on Ch.25 of silkworm, based on silkworm precise mapping and tissue chip data of whole genome. The main findings follow:1. The cloning and expression profile of bHLH gene in silkwormAccording to the data of precise mapping and the tissue chip data of whole genome, it is found that 2 bHLH genes expressed in silk gland by Liu C. The expressed bHLH genes in silk gland, the probe are sw15083 and sw01142, the gene ID are BGIBMGA005127 and BGIBMGA007303. According to the homologous to sage and dimm gene in fruit fly, named Bmsage and Bmdimm. Most of the known transcriptional and regulation factors belong to homedomain families. The discovery of bHLH gene in silk gland is great significant to regulation of silk gene expression. Bmsage gene is on CH.25th, the gene length is 2.9kb, the total length of mRNA is 1199bp which includes a completed 720bp of coding frame that predicts to code 234 amino acids. This gene is single copy gene, made of 4 exons and 3 introns; there is a core promoter in the upstream(-699-650) of transcription initiation site. The upstream of the promoter contains basic elements:-10 region and CAAT frame; there are two polyA termination signals in the downstream of 3' terminal.Bmdimm gene is on CH.17th single copy gene, made of 4 exons and 3 introns. the gene length is 4.6kb, the total length of mRNA is 1,593bp which includes a completed 636bp of coding frame that predicts to code 211 amino acids. There is a core promoter in the upstream(-1865～-1914) of transcription initiation site. The upstream of the promoter contains servral basic elements:-10 region and CAAT frame; there are not polyA termination signals in the downstream of 3'terminal.It is seldom reported about the bHLH-class transcriptional factors. Sage gene of fruit fly regulates salivary gland-specific expression. Salivary is recognized as the most possible homologous tissue, which is similar characteristic of silk gland. In the in situ hybridization experiment, it is demonstrated that Bmsage gene is expressed in silk gland nuclei during the day 5 of fifth in star. Bmsage gene is up-regulated in the intermolt period, and down-regulated in the molt period and metamorphosis. Bmdimm gene is expressed specifically in silk gland, Bmsage gene is up-regulated in the 4th instar molt period and previous period of 5th instar, and down-rcgulated in the later period and metamorphosis. It is found that there are many bIILH binding sites in the FibH gene introns, which are adjacent to the binding site of FMBP-1, a transcriptional factor. It is speculated that Bmsage gene is important to regulate the expression of FibH gene.2. Study of the FibH promoter expression mediated by AcMNPV in the silkworm tissuesFibroin gene is a important structural gene in the silk gland of silkworm. The mRNA of FibH gene is expressed in posterior silk gland. The expression of FibH gene is accumulated in the inter-molt period, decreased in the molt period. FibH gene is closed in the molt period, in which the early accumulated mRNAs are degraded. In order to analysis the new cis-regulatory domain of FibH gene in silkworm, three 5' terminal-truncated FibH promoters that are different from sizes has been cloned. The recombinant Autographa california multiple nuclear polyhedrosis virus (rAcMNPV) are used as gene transfer vectors. It is found that three FibH gene promoters effectively expressed, mediated by virus, the exogenous genes in silk gland, fat body and hemolymph, which the fluorescence is detected on the surface. It is suggested that the tissue-specifically expressed feature of FibH promoter changed. In contrast experiment, Serl promoter still specifically promotes to downstream gene expression in the posterior silk gland. Compared the expression of different FibH promoters in size,0.2K and 0.9K of promoters express genes in the middle and posterior silk glands, but 2. 1K of promoter is express genes only in the posterior silk gland. The ectopic expression of FibH promoter in the silkworm tissues, we speculate, is probably because that there are many transcriptional enhancement elements in 5'upstream of FibH, which are enhanced the downstream gene transcription after recognized by other cell factors. FibH is integrated into the virus genome. Some of the elements in virus itself probably affect the activity of FibH promoter. Hence, it leads to ectopic expression. The gene expression or maintenance of some specificity needs specific circumstances. The normal physiological and biochemical reactions of silkworm are influenced by virus infection. The micro-environment of cells is also affected. Thus, it changed the original feature of FibH gene. After virus infection, the change of environmental condition makes FibH promoter ectopically express genes in fat body and hemolymph.3. The influence of FibH promoter by AcMNPV infectionThe early research suggests that FibH gene is specifically expressed in the posterior silk gland. It is specifically synthesized in the posterior silk gland during the inter-molt period, but the expression is inhibited during the molt period.FibH promoter has the phenomenon of ectopic expression, if AcMNPV is as the gene transfer vector. In order to detect that whether AcMNPV affects FibH promoter or not, we infected FibH-transgenic silkworm, using donor AcMNPV. It is found that the expressed position of EGFP did not change, is still expressed in the silk gland. After eliminating virus infection, the up-regulation of some of unexpressed genes activated, and these genes further activated the expressed probability of FibH in other tissues. Meantime, it is not the results that virus-specific expressed genes influence the activity of promoter. In recent years, it is reported that fibroin gene has the phenomenon of leakage transcription in other silkworm tissues. p25 gene not only transcribes effectively in the posterior silk gland, but also transcribes in other tissues, such as ovarian tissue, during the larva period. There are different transcriptional initiation sites in silk gland and ovary. Three types of fibroin protein genes infected BmN cells, which is expressed, to certain extent.4. Speculation the reason of protein ectopic expressionCombining our research, it is speculated that the FibH promoter is integrated into the virus genome, some cis-elements of virus itself affect the activity of FibH promoter, which change the original features. The transcription of FibH gene promoter is not strict, the transcription in other tissues also exists. Due to the virus large reproduction, the contents of FibH promoter in silkworm tissues is much more than that in the normal condition. The accumulation of a little transcription ultimately produces the phenomenon of protein ectopic expression, which is able to be detected. The speculation needs to be the experimental support of FibH gene expression and regulation research. Although, it is reported that FibH gene exists leakage phenomenon in the middle silk gland and other tissues, it is not found that the silk protein is expressed in tissues except from silk gland. The genes in the silk gland have strict tissue specificity. This shows the transcriptional and translational regulation of FibH gene is more complicated than that humans think. It is necessary to do further research in order to illustrate the regulation mechanism.