Cloning And Effect Of Exogenous Factors On The Expression Of Pkchs Of Pinus Koraiensis

Chalcone synthase (CHS; EC2.3.1.74) is the key enzyme of flavonoidbiosynthesis. It catalyses the stepwise condensation of4-coumaroyl-coenzyme A (CoA)derived from the phenylpropanoid pathway with three acetate moieties derived frommalonyl-CoA to give rise to the C15flavonoid skeleton, naringenin chalcone.Isomerization and further substitution of this central intermediate lead to the synthesisof flavones, isoflavonoids and anthocyanins. In accordance with the specializedfunctions of the various types of flavonoids, the activity of CHS is specifically regulatedby different external stimuli or by endogenous mechanisms in plant development andtissue differentiation. This study mainly studies the Pinus koraiensis，the main pinespecies in northeast China. Cloned the chalcone synthase gene PkCHS from theadventitious bud of Pinus koraiensis by RT-PCR method, and study of variousexogenous factors on its expression. Providing a theoretical basis for scientific andreasonable development of pine polyphenols.Extract RNA from adventitious bud of pinus koraiensis. Synthetise the first-strandof cDNA by reverse transcription，then using PCR for amplification. Recycling PCRproducts which contain the objective genes, and linked it to the pGM-T Vector, thentransform it into TOP10cell. Extracting positive cloning plasmid, PCR amplification.Deliver the positive clone to sequencing. Results show that the length of cDNAsequence of the PkCHS gene is1188bp and it encodes395amino acids. The results ofgene homology analysis show that the PkCHS and CHS of Pinus strobus have thehighest homology, which is98.74%. Amino acids homology analysis results suggestthat, the homology of PkCHS with CHS of Pinus strobus is the highest too, whicharrived at98.79%.According to the results of exogenous factors influence on pinepolyphenol content, glucose could inhibit the synthesis of pine polyphenols, while lowtemperature and raffinose could promote the synthesis of pine polyphenols. Applicationof real-time fluorescent quantitative PCR analysis of PkCHS on gene expression, showthat glucose could inhibit PkCHS gene expression, while the low temperature andraffinose could facilitate PkCHS gene expression.