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(Insertion & Expression Of Foreign Genes In Transgenic Silkworm, Bombyx Mori L.)

Posted on:2001-02-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:X ChenFull Text:PDF
GTID:1103360002952451Subject:Special economic animal breeding science
Abstract/Summary:PDF Full Text Request
In recent years, the transgenic animal research has been becoming one of the best hot-points in Genetics and Biology. Silkworm (Bombyx mon. L.), not only is an important economic insect, but also is one of important model organisms for researches on classical genetics. Studies on transgenic silkworm possess theoretical significance and practical value. It抯 also an extremely powerful tool for investigating the mechanism related to gene regulation and protein function, etc. There are mainly three parts in this thesis, they are: (1) Neomycin resistance transgenic silkworm We, first time, attempted t e possibilities on using the neomycin resistance gene (Ned?) as a positive selectiv marker in transgenic silkworm studies A transport plasmid pFN was constructe , in which the NeoI? was controlled by BmNP V-lit (Born byx mon Nuclear Polyhe rosis \lirus immediately early stage gene, BmNP V-fE) promoter and flanked with 5 and 3?homogenous regions of fibroin heavy-chain gene of silkworm. This plasm d was linearized and transformed into Bin-N cells, as well as silkworm eggs in the early period of fertilization by gene gun. In vivo, the Ned? Bin-N cell lines can be obtained by 0418 selection, and in vitro, the NeoR transgenic silkworm can be obtained by rearing selection of the new hatched larva with artificial diet for 48hrs, in which 8mg/ml neomycin was added. PCR and Southern blotting analysis for Ned? in (12 and G4 genomic DNA showed that, the NeaR sequence had been integrated into the neomycin resistant silkworm chromosomes. Our result also suggested that the Neal? is an efficient selective marker in transgenic silkworm study. (2) Ribozyme mediated anti-NPV in transgenic silkworm Ribozymes are small catalytic RNA molecules. Silkworm Nuclear Polyhedrosis sepsis caused by BmNPV is one of grievous silkworm diseases in practice. To establish a gene therapy system for BmNPV, a triplet hammerhead ribozyme pBNRz426 was designed and synthesized, which targets three different sites in BmNPV IE mRNA, due to the IE acts an important function role in NPV original replication. A recombinant expression plasmid plE-Luc-Rz containing pBNRz426, IE promoter and Luciferase was constructed. After confirming the triplet ribozyme was efficient in vivo, we attempted to integrate the pIE-Luc-Rz into silkworm chromosome. First, the pIE-Luc-Rz digested by ScaI was transformed into silkworm eggs in the early period of fertilization by gene gun. Second, selected the positive individual in G 1 generation by analysis of * Luciferase activities. At last, inherited the anti-NPV transgenic silkworm by infecting the offspring with BmNPV. Thus, we obtained the ribozyme-mediated anti-NPV transgenic silkworm line-- AF2. PCR detection and Southern blotting of G4 gDNA showed that the synthesized ribozyme gene and the reporter gene are all integrated into transgenic chromosomes with many copies. Meanwhile, the triplet ribozyme can be detected in different ? developmental stage and all tissues in silkworm by RT-PCR amplification. Northern blotting was also used to detect the expression of ribozyme. In vitro cleavage by ribozyme separated from total RNA of transgenic pupa showed that the ribozyme was still active and could cleave the viral mRNA site-specifically as in vivo.
Keywords/Search Tags:Transgenic silkworm, NeaR, Ribozyme, Anti-NPV, Gene targeting
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