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Cloning and Transformation of the Key Enzyme Gene(4CL) cDNA in Resveratrol Synthesis with Saccharomyces Cerevisiae

Author: WangYongZhi
Tutor: WeiYaHui
School: Northwestern University
Course: Cell Biology
Keywords: Resveratrol 4 - coumarate : coenzyme A ligase Saccharomyces cerevisiae Shuttle vector
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 153
Quote: 1
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Abstract


Resveratrol is a phytoalexin , has antibacterial, antioxidant protection cardiovascular, cancer and many other pharmacological effects . Now widely used in food, medicine , health care products and other industries . Production resveratrol main plant extracts , under natural conditions , plants resveratrol content is very low , limiting the production of resveratrol . Therefore, the use of modern biotechnology to produce resveratrol attention has been paid gradually . Saccharomyces cerevisiae with fast growth , require low to medium production process does not produce toxins and other advantages , the use of recombinant Saccharomyces cerevisiae production Resveratrol is an effective way . However , another key enzyme in the absence of resveratrol biosynthesis in Saccharomyces cerevisiae in vivo , 4 - coumarate : coenzyme A ligase ( 4CL ) . It should first 4CL gene by means of genetic engineering transformed into Saccharomyces cerevisiae cells . In this study, The TRIzol method Arabidopsis total RNA by RT-PCR Cloning 4 - coumarate : coenzyme A ligase gene ( 4CL ) cDNA sequence , and construct the recombinant shuttle vector pESC - HIS -4Cl . With pESC-HIS-4CL into E. coli DH5 , screened by ampicillin-resistant Escherichia coli . Extraction of recombinant Escherichia coli plasmid by PCR and restriction enzyme digestion and sequenced determination and sequencing results and NCBI accession number NM 0 < / Sub > 01,084,228.1 4CLcDNA sequence alignment , homology was 99 % . Prove the 4CL gene has been successfully cloned into pESC - HIS vector . Lithium chloride, the recombinant shuttle vector pESC-HIS-4CL was transformed into S. cerevisiae transformants screened by SD histidine auxotrophic medium . Extracted plasmid transformants , identified by PCR and restriction analysis results show that the success pESC-HIS-4CL was transformed into Saccharomyces cerevisiae .

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