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Cloning and Functional Analysis of BmDichaete Gene in the Silkworm, Bombyx Mori

Author: PengLiNa
Tutor: LuCheng；PanMinHui
School: Southwestern University
Course: Cell Biology
Keywords: Bombyx mori Sox genes BmDichaete Embryo development RNAi
CLC: Q78
Type: Master's thesis
Year: 2012
Downloads: 32
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Abstract

Sox genes, as important transcriptional factors, abundantly involve in most of developmental processes. Sinclair et al identified SRY/Sry (sex determining region of Y chromosome) gene in human and mice in1990. From then on, a series of homologous genes encoding transcription factors have been found collectively referred to as Sox (Sry-related box) genes. Sox genes encode a class of transcription factors with a high sequence similarity to sex-determining gene Sry in animals. The family is characterized by the presence of the conserved high-mobility group (HMG) box. Studies on fruit fly, mouse and other model organisms have shown that Sox gene family plays important roles in regulating sex differentiation and development, gonad development, early embryogenesis, nervous system development, hematopoiesis and the formation of cartilage and other tissues and organs.The silkworm, Bombyx mori, as a model of lepidopteral insects, is an economically important insect. There is little research on Sox genes in the silkworm. Dichaete gene, which belongs to B subgroup of Sox gene family, was first found in Drosophila, and is the orthologue of vertebrate Sox2. As a transcription factor, Dichaete gene has extensive and important roles in the specification and development of Drosophila nervous system. It also plays critical role in the development of other tissues, such as hindgut development, oocytes formation. Based on the advanced research achievements on Sox genes in fruit fly and other species as well as the information of silkworm genome sequence, we cloned the homologene of Drosophila Dichaete in silkworm, named BmDichaete. In addition, we explored the gene expression pattern and functions using RT-PCR and RNAi. The main results are as follows: 1The information analysis and full-length cDNA cloning of BmDichaete gene in silkwormThe bioinformatics analysis shows that BmDichaete gene is located in nscarf2589on chromosome4of silkworm. The full-length cDNA of BmDichaete gene was obtained by RACE approach. It contains1541bp encoding for a protein with246amino acid residues. The length of ORF is741bp, with only one exon and no introns. The predicted protein weighs27.3kD, and isoelectric point9.89. Bioinformatic analysis suggests that BmDichaete protein sequence contains a typical HMG-box domain. Homology analysis shows that BmDichaete gene is highly conserved in evolution. BmDichaete gene and its homologies in other species share90%similarity in HMG-box domain. They also have some conserved sequences out of the HMG-box. Sox genes in silkworm share about50%conservation in HMG-box, but no sequence similarity outside the HMG-box.2Prokaryotic expression of BmDichaete geneGiven that BmDichaete gene shares90%sequence similarity in the HMG-box domain with other members in the same group, we cloned two segments in vitro:one is the complete ORF, and the other is the specific sequences except the conserved domain. The target sequences were subcloned into the pET-28a-c(+) expression vector, then transforming the recombinant plasmid into Escherichia coli BL21. The exogenous protein was induced by IPTG. Finally, we got two recombinant proteins with molecular weight about32.3kD and17kD, including a six-His tags protein, which is consistent with prediction. The proteins are expressed as inclusion bodies by appraisal. IPTG concentration and induction time have little effect on protein expression, but induction temperature can greatly affect the target protein expression level. The optimal condition of protein expression is0.4mM IPTG,37℃induced5hours through optimization.3Preparation of BmDichaete antibodyTarget protein was purified by affinity chromatography and then used to produce specific antibody. The antibody titer is1:100000detected by indirect ELISA.4The expression pattern of BmDichaeteRT-PCR analysis shows that BmDichaete gene expresses through the entire development of silkworm. The expression level reduced during the late pupal stage and very little in moth. BmDichaete also shows tissue-specific expression pattern during larval stage. It’s expression mainly restricted in the nerve system, brain, ovary, hindgut and silkgland. Western blotting analysis shows an obvious27kD bands, which is consistent with the RT-PCR results.5RNAi of BmDichaete during silkworm embryogenesisWe silenced BmDichaete gene in the developing embryo of silkworm by dsRNA-mediated RNAi. Through observing the turning-green rate and hatchability of the injected eggs, also observing the internal structure of the embryo by paraffin section, we didn’t find obvious difference between the injected eggs and the control group.

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