Cloning And Transformation Of The Key Enzyme Gene(4CL) CDNA In Resveratrol Synthesis With Saccharomyces Cerevisiae

Resveratrol, which is a phytoalexin, has many pharmacological effects such as antibacterial effect, antioxidation, cardiovascular protecting effect, anti-cancer effect and so on. It has been widely used in food, medicine and health protection industry. The method of getting resveratrol is mainly extracting from plants directly at present. However, the content in plants is so low that the output is greatly restricted. Resveratrol's producing with modern biotechnology has gradually been rewarded much attention. It is a good method to use yeast recombinant to produce resveratrol due to Saccharomyces cerevisiae's rapid growth, little bias on culture medium and no toxin production. But Saccharomyces cerevisiae lacks the key enzyme (4-Coumaric acid Ligase, 4CL) in resveratrol biosynthesis. So firstly 4CL gene must be transformed into Saccharomyces cerevisiae cells with gene bioengineering means. The main procedures are as follows:1 getting arabidopsis thaliana's total RNA with TRIzol method2 cloning cDNA sequence of 4-Coumaric acid Ligase (4CL) with RT-PCR method3 constructing recombined shuttle vector pESC-HIS-4CL4 screening recombined E.Coli though ampicillin replication experiment after transforming DH5αof E.Coli with pESC-HIS-4CL5 extracting the plasmids of the recombined E. Coli6 comparing the sequence identification results with 4CLcDNA sequence of NM₀01084228.1(accession number in NCBI)7 transforming the recombined shuttle vector pESC-HIS-4CL to Saccharomyces cerevisiae with Lithium Chloride( LiCl) method8 screening the transformant using SD medium (His nutritional Deficiency culture, SD)9 screening the plasmids of the transformant and identifying the results through PCR and Double DigestsThe result of step6 shows that 4CL gene has been successfully transformed into pESC-HIS vector with 99% homology. And pESC-HIS-4CL has already been transformed into Saccharomyces cerevisiae after step9...