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Molecular Cloning and Primary Functional Analysis on Gene CpABC from Chimonanthus Praecox (L.) Link

Author: WangXianLei
Tutor: LiMingYang
School: Southwestern University
Course: Ornamental Plants and Horticulture
Keywords: ABC transporter protein Lamei Clone Vector construction Expression Analysis
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 43
Quote: 1
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Abstract

ABC transporters (ATP-Binding Cassette transporter, ABC transporter) is a widely found in prokaryotes and eukaryotes super family protein function has been studied most widely, a protein in the largest number of It primarily as a transmembrane protein present in the biofilm on. In recent decades, it was discovered that the ABC transporter protein in many organisms (including prokaryotes and eukaryotes) will participate in a number of important physiological processes, such as a secondary product of the metabolic processes of plants, bacteria resistance process, even the human body resistance of some tumor cells have very close contact, which has important research value, people began widespread concern and research the ABC transporter protein. Wintersweet (Chimonanthus praecox (L.) Link) is native to China, Tertiary relict plant and China National two rare and endangered plants. Bud flowering December to next early spring in February, at the beginning of winter frost cold days, proudly opened in windy Shuangfeng typical winter flower plant, known as the \three noes \An ABC transporter gene CpABC the papers from wax Plum organ cloning and expression in E. coli, and then analyze the expression products, and achieved major results are as follows: 1. Wintersweet ABC transporter gene cloning selection of Wintersweet cDNA by random the library sequencing won size of the a 707bp EST sequences, and then cloned the full-length cDNA by hiTAIL-PCR, RACE technology. The BLASTX than the results show that with the ABC transporter protein having a high homology, the preliminary estimation of the obtained cDNA encodes the ABC transporter protein, named CpABC. CpABC cDNA full-length 2818bp, which contains an open reading frame of 2145bp encoding protein length of 714 amino acids. 2. Plant expression vector construction box CpABC ORF fragment pMD-19T carrier, then with BamHI and SacI double enzyme Qieke Long vectors and expression vectors pCAMBIA2301g recycling target fragment and expression vector and connect by restriction enzyme digestion and PCR Verification successfully constructed expression vector containing the gene. 3 Construction of prokaryotic expression vector design primers with the BamW I and SacI digested sites, PCR of C. praecox CpABC gene was amplified with BamW I and SacI the double digestion Wintersweet CpABC gene and prokaryotic expression vector PET-28a, and then the the genes connected Wintersweet CpABC After digestion, PCR validation and sequencing showed that the prokaryotic expression vector containing the gene has been successfully constructed prokaryotic expression vector PET-28a. The 4.CpABC induced expression of genes in E. coli will build good prokaryotic expression vector into E. coli, through appropriate inducing conditions induced CpABC gene expression in Transetta (DE3) strain, and recombinant proteins by SDS-PAGE. proved gene expression CpABC. Expression Analysis of 5.CpABC gene CpABC expression in flower buds of the least amount of almost no expression in the bud period with the increase but rarely exposed flap during the higher expression level and the early opening, bloom period in turn reduce, and finally in The decline during the highest expression level. Highest expression level in the CpABC outer flap, and then from the flap within petal, stamen, pistil turn reduce this display CpABC with Wintersweet growth activities are closely related.

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