Genetic Transformation Of Yeast With 4CL CDNA Cloning From Arabidopsis Thaliana

In this study,the total RNA was abstracted from Arabidopsis thaliana,and then gene of 4-coumarate:CoA ligase was cloned by RT-PCR.After that the cloning vector pGEM-TEasy-4CL was constructed.After the sequence was determined,the 4CL gene was subcloned into the expression vector pPIC3.5K to yield the recombinant expression vector pPIC3.5K-4CL.The recombinant plasmid was transformed into P.pastoris GS115 by electroporation.The main achievements of this research were as follows:The 4CL gene were amplified from Arabidopsis thaliana by RT-PCR.The 4CL gene was ligated into the pGEM-TEasy vector.The recombinant vectors were assayed by restriction digestion and sequencing.Then the sequence was blasted with the 4CL cDNA titled NM₀01084228.1 in the NCBI.And the homology was 99%.The results showed that the 4CL gene was successfully cloned and ligated into the cloning vector pGEM-TEasy.For constructing the expression vector pGEM-TEasy-4CL,two primers of 4CL gene with BamH I and Not I site respectively were designed.Then the 4CL gene with the two restriction enzyme sites at its 5' end and 3' end respectively was amplified and inserted into the expression vector pPIC3.5K.The recombinant expression vector pPIC3.5K-4CL was transformed into DH5αand identified by PCR and restriction enzyme BamH I and Not I. The results indicated that the gene was correctly cloned into the vector.The expression vector pPIC3.5K-4CL was transformed into P.pastoris(GS115)by electroporation.Many recombinants were obtained after a series of screening,including auxotroph,G418,PCR.