Dissertation > Excellent graduate degree dissertation topics show

The Construction and Transform of Rice Expression Vector of CTB Gene and CTB-ureB Fusion Gene

Author: JiangYunShui
Tutor: FangPingChu
School: Zhejiang University
Course: Pathogen Biology
Keywords: Transgenic Plants Hp ureB gene CTB gene Rice expression vector Fusion gene
CLC: R346
Type: Master's thesis
Year: 2004
Downloads: 110
Quote: 0
Read: Download Dissertation

Abstract

Since the advent of the first genetically modified plants in 1983, the rapid development of plant genetic engineering, has made remarkable achievements in plant virus resistance, insect resistance, herbicide resistance, stress tolerance improved varieties and exogenous gene expression, transgenic plants as a cheap bioreactor production of human pharmaceutical materials show attractive prospect. Rice is the most important, the most extensive range of planting food crops in the world. Today, as the representative of Gramineae rice transgenic technology has made a major breakthrough, Rice expressing heterologous proteins become possible. Rice through transgenic technology, expression systems produce safe and cheap genetically modified vaccines, antibodies, and even cell factor, not only the progress of medicine genetic engineering research, also the rice from simple food crops into functional foods with high added value an important way. Helicobacter pylori (Helicobacter pylori, Hp) is a major virulence factor of chronic gastritis and peptic ulcer by WHO as gastric cancer is closely related to pathogens considered the class Ⅰ human carcinogen (Group I carcinogen). The use of the vaccine is the most effective method for the prevention of Hp infection. This study by plant transgenic technology, will the Hp ureB gene and adjuvant CTB gene into rice chromosome DNA and stable inheritance of the regenerated plants expressing microbial antigens Hp transgenic plant vaccines bioreactor for the establishment of rice laid foundation. Method 1. Rice expression vector p13W4-CTB: building CTB gene to pGEM-CTB as a template, PCR amplification CTB gene, BamHI digested the carrier p13W4 and the dephosphorylation, and then connected with the same digested the CTB PCR product, PCR screening, restriction digestion Identification of positive clones, DNA sequence analysis verified. 2. Build The CTB-ureB fusion gene rice expression vector pCWUN: (1) CTB, ureB gene fusion and cloning: high fidelity PCR technology the CTB and cagA gene was amplified from plasmid pGEM-CTB and Helicobacter pylori genomic DNA , then fusion of the two genes by the PCR method, and finally the CTB-ureB fusion gene was inserted into the vector pUC19, the PCR screening digestion positive clone was identified, DNA sequence analysis verified. (2) rice Wx promoter transformation: using high-fidelity PCR techniques, Wx promoter was amplified from plasmid p13W4 the ΩK5 'and 3' primer 72 ° C annealing extension product was obtained OK fragments, two fragments were then fused by PCR method obtained Wx QK fusion fragment. (3) the CTB a ureB fusion gene in rice expression vector pCWUN: the PCR synthesis NoS terminator, from the cut plasmid PUCU the CTB a ureB fusion gene, followed by the NOS terminator, the CTB a ureB fusion gene and Wx an OK fusion fragment insert the plasmid PCAMB recognize 1300, screening, the recombinant plasmids PC share Jidao bite. Rice expression of of carrier P13w4 a cTB and pcWIJN were transformed into competent state tumefaciens tumefaciens transformants obtained after PcR Screening and restriction enzyme digestion. Rice immature embryo induction of embryogenic callus and Agrobacterium tumefaciens-mediated expression vector P13W4 C transformation of rice embryogenic callus resistance screening, differentiation and rooting culture of transgenic plants, PCR identification of transgenic plants. PCR analysis, restriction endonuclease and its DNA sequence analysis confirmed that the the CTB gene has been inserted into the carrier P13W4 the right direction, and the cloned gene reported in the corresponding nucleotide acid sequence homology of 100%; NOS terminator sub , CTB ~ ureB fusion gene and W Bu. K fusion fragment in the correct orientation, the corresponding sequence is inserted into the vector pCAMBIA1300 CTB ureB fusion gene in correct reading frame, to compare it with the corresponding gene sequences published in GenBank, the the CTB gene sequence homology is 100%, The deduced amino acid sequence homology is 100%. Speak eB gene sequence homology% .0% 97.7% 98.8% 99.5% amino acid homology. 2.PcR analysis, restriction enzyme digestion confirmed the rice expression of of carrier P13W4 a cTB and pCWUN respectively, successfully transform competent Agrobacterium tumefaciens. 3.PCR analysis confirmed the CTB gene has been integrated in the chromosomal DNA of the rice seedlings. Conclusion 1. Successfully constructed a rice expression vector pl3W4 CTB and pWCUN of Sequence analysis confirmed that the sequence is correct. Rice expressing the carrier p13w4 a CTB and pwcUN successful transformation of Agrobacterium tumefaciens. 3. Success of Agrobacterium tumefaciens-mediated p13W4 CTB transformed rice transgenic plants.

The Construction and Transform of Rice Expression Vector of CTB Gene and CTB-ureB Fusion Gene

Abstract

Related Dissertations