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The Thrombolytics Gene Cloning And Vector Construction And Mefenacet Dryland Plant Dna Interaction Studies,

Posted on:2004-09-22Degree:MasterType:Thesis
Country:ChinaCandidate:H HuFull Text:PDF
GTID:2191360125958552Subject:Applied Chemistry
Abstract/Summary:PDF Full Text Request
1. The plasminogen activator can activate the plasminogen to form the fibrolase which can catalyze hydrolyzation of the fibrin. We will transfer the fibrolase gene into plants by the transgenic technology to product the fibrinoclic medicine by "plant reactor". In this paper,the plant recombinant expression plasmid of fibrolase gene pBI121-zzq was constructed by subcloning fibrolase gene into shuttle vector pBI121 that contains NPTII encoding kanamycin resistance. The plasmid pBI121-zzq was then transformated into E.coli DH5a and A.tumefaciens LBA4404. The transformants were screened from kanamycin antibiotic plate. The recombinant plasmid DNA was isolated by the alkaline extraction method and detected by the agarose gel electrophoresis. On the agarose gel plate, a band of about 200bp appears, its size was as same as the target gene, which proved the target gene had already introduced into the bacterium. The successful construction of the plant expression vector pBI121-zzq is the base of the transfermation of the plants and the exploitation of the novel iatric plants.2. The interaction of 2-(2-benzothiazolyloxy)-N-methyl-N-phynyl-acet -amide(BTMPA) with maize DNA and setaria DNA was studied by UV spectrophotometry, and a vovel grass-removal mechamism was recommend -ed. The BTMPA can untwist the DNA helix to denature the grass DNA, which inhibited the replication of DNA. So the setaria was eliminated.(1). The conditions for extraction of maize DNA and tomato DNA were optimized. The optimum conditions were as following (for 1.0g sample): 4.0ml of the cell extraction solution, 1.5ml of KC1 saturated solution, the rate between the phenol/chloroform/iso- pentanol solution, chloroform/iso-pentanol solution and isopropyl alcohol and the sample solution being 0.8,0.7 and 0.9, respectively, the proportion between the phenol and chloroform/ isopentanol was 24:25:1. Under the optimum conditions the extracted DNA was very pure, and the yield was 110 g/g.(2). The optimum conditions for the extraction of setaria DNA were as following (for 1.0g sample): 4.0ml of the cell extraction solution, 1.5ml of NaAc solution, 60 minutes of heat time at 65 C, the proportion between the phenol and chloroform/ isopentanol was 26:23:1. The rate between the phenol/chloroform/ isopentanol solution, chloroform/isopenta-nol solution and isopropyl alcohol and the sample solution being 0.1, 1.0, 1.0 and 1.0, respectively. Under the optimum conditions the extracted DNA was very pure, and the yield was 750 g/g.(3). A novel grass-removal mechanism is first discovered. The BTMPA can denatured the setaria DNA and inhibit the replication of the DNA, so the setaria was eliminated. For BTMPA concentration less than 5. 4 x 10-9mol/L the BTMPA could not interact with setaria DNA, and for larger than 1. 8x 10"4mol/L the BTMPA can interact with maize DNA. So the optimum concentration using BTMPA for elimination of setaria was from 5. 4 10-9mol/L to 1. 8 10-4mol/L, and the optimum temperature was 20-30 C.
Keywords/Search Tags:fibrolase gene, expression vector, transformation, DNA extraction, optimize, BTMPA
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