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The Establishment Of High Frequency Transformation System Of Tomato And The Studies On Transgenic HBMP-3m Gene In Tomato And HBMP-3 Gene In Tobacco

Posted on:2004-05-08Degree:MasterType:Thesis
Country:ChinaCandidate:X G ZhaoFull Text:PDF
GTID:2120360095450574Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Human bone morphogenetic protein 3 is a member of TGF- B superfamily.lt can induce the differentiation of cartilage and bone tissue in mesenchymal cell.and is important to bone self-repairment and bone development during embryo morphogenesis.In addition,some other biological activities of hBMP-3 have also been found.such as inducing development of embryo and stimulating differentiation of neural and blood cells.Therefore,there is a great prospect in the use of hBMP-3.There is trace content of hBMP-3 in human body.It has been expressed in the expression system of eukaryotes and prokaryotes respectively,but its application is restricted because of defects in the process and modification after translation in prokaryotic cells and higher costs and lower yields existed in eukaryotic expression system. With the successful expression of exogenous gene in plant.the advantage of plant expression system become a highlight increasingly.Based on the successful expression of hBMP-3m gene into tobacco,we maked the study of transferring hBMP-3m gene into tomato and hBMP-3 gene into tobacco ,in order to obtain tomato transgenic plant with hBMP-3m gene and tobacco transgenic plant with hBMP-3 gene, to establish basis for getting step farther of leaning the expression of hBMP-3m gene and hBMP-3 gene in plant,the difference between the product of hBMP-3m gene and hBMP-3 gene in plant and the active of expressible product.Based on the plant expression vector of pCAMBIA1300-hBMP-3m and pCAMBIAl 301-hBMP-3 constructed,we set up the system of high frequency regeneration and transformation of tomato cotyledons.The hBMP-3m gene and hBMP-3 gene were respectively transformed into tomato and tobacco by transfection of Agrobacterium tumefaciens.And the transgentic plants were detected.The main results are as follows:l.The plasmid pCAMBIAl300-hBMP-3m carried hBMP-3m gene and pCAMBIA -1301-hBMP-3 carried hBMP-3 gene were respectively transferred into Agrobacterium tumefaciens by electroporation.2.According to the effect of combination of different hormone concentration in the medium on callus formation and shoot induction of tomato cotvledons,we defined MSo+2.0mg/L BA+-0.2mg/L IAA as optimum differential medium.3.Through the ladder experiment of selecting transformer concentration by Hyg, the proper selecting transformer concentration is 20mg/L Hyg to tomato cotyledons and 35mg/L Hyg to tobacco discs.4.After having established genetic transformation system with tomato cotyledons as explant and determined the transformable of preculture time,incubation time and co-culture time,we set up the system of high frequency transformation of tomato cotyledons.Then hBMP-3m gene was transferred into tomato via Agrobacterium-mQdiated transformation,and the resistant plants to Hyg were obtained.By PCR analysis on part of the putative transformants,we identified that hBMP-3m gene had been integrated into the genome of part of tomato plants.5.Transferred hBMP-3 gene into tobacco via Agrobacterium-mediated transformation and obtained the resistant plants to Hyg. Trans genie tobacco plants were confirmed by instantaneous expression of GUS gene in calli detection,growth and bio-morphology analysis,Hyg-resistant experiment and PCR analysis.
Keywords/Search Tags:Human Bone Morphogenetic Proteins 3 gene, Lycopesicum esculentum Mill. Nicotiana tabacum, Agrobacterium tumefaciens mediation, Regeneration system.Genetic transformation.
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