Gene Splicing In Vitro Using A Nuclear Extract From Bombyx Mori Fat Body

Splicing and regulation of pre-mRNA are complicated processes, which involve many genes in advanced eukaryotes. It plays an important role in correctly functioning of genes. Splicing of pre-mRNA, namely removing introns and assembling exons into mature mRNA, is one of important taches in genic expression and regulation. selective splicing is a common affair in multicellular organisms , it exists in about thirty percent of human's genes.The function, behaviours and their own complexity of multicellular organisms evolved through increasing albumen diversity or effiectively harmonizing the network relationship of elements.The silkworm was an important economic insect.furthermore,it was one of the model creatures in classical genetics. Many studies showed male silkworm had higher resistance, transformation efficiency and better constitution than female. only raising male silkworm would increase the silk-production by 15% without no augment of cost. Meanwhile,silk produced by male silkworm was fit to produce high-rank raw silk due to its thin intensity ,little windage and good quality. therefore, studying the sex-determination mechanism of silkworm would finally implement the raising of male silkworm and effectively increase producing efficiency of sericulture and economic benefit. Simultaneously, lepidoptera was a major kind of pests silkworm was a typical representative in Lepidoptera. Clarifying the sex-determination mechanism would contribute to the studies on control of insects .In the mechanism of sex-determination in silkworm, the selective splicing play a important role.Bmdsx would produce sex differentiation due to its different splicing modes.. Experiments demonstrated Bmdsx ,as the same as dsx in drosophila. which had different processing fashion in male and female, was a tolerant splicing mode in female silkworm.but in male ,it was barrier splicing: namely one barrier blockaded the splicing between the third and forth exons, thus the two exons did not exist in male. In order to investigate the effect of upstream barrier on the spicing of Bmdsx, we constructed splicing system in vitro.First, we constructed splicing system in vitro by RAB4B.then,We carried out studies of Bmdsx on splicing in vitro in nucleolus of fat body in female silkworm. RAB4B was a apoptotic gene with a size of about 1400bp.thereinto, Exon was 1200bp and intron was 200bp . because intron in Bmdsx was relatively large, it was difficult for Bmdsx to splice in vitro. it was in need to construct a mini Bmdsx gene to remove the majority of introns and only leave smaller introns essential to splicing.The mini Bmdsx was 2264bp,containing 4 exons and 3 introns. The gene cloned into vector pGEM-T Easy was purified after PCR amplification then transcriped in vitro. The pre-mRNA spliced in vitro in nucleous abstract. when the mini Bmdsx already constructed was spliced in male nucleous abstract,the third , fourth exons and the introns were cut together,while the second and the fifth exons linked together ,with a size of 362bp.Bmdsx played an important role in the sex-control mechanism of silkworm constructing a splicing system of mini Bmdsx contributed to the studies on sex-differentiation. through selective splicing in female and male , Bmdsx ultimately determined individual sex differentiation .the study established a splicing system of mini Bmdsx in vitro,which was instrumental in the studies on splicing system of Bmdsx through carring out splicing experiments in vitro and looking for regulatory factors located in the upstream of Bmdsx and clarifying the molecular mechanism in sex determination of silkworm.