The Construction Of Expression Vector And Genetic Transformation Of4-coumarate:CoA Ligase In Betula Platyphylla Suk

Lignin is a kind of macromolecular organic matter with very rich content in plants, which is second only to that of cellulose. It is involved in the formation of plants secondary cell wall, necessary in vascular plants growth and development and a very important factor in plants evolutionary process.4-coumaric acid coenzyme A ligase(4-coumarate:CoA ligase,4CL) participate in the synthesis of lignin, plant antitokin and flavonoids, etc, as a key enzyme in phenylpropanes metabolic processes. In lignin biosynthesis,4CL gene play a linking role in the association between lignin precursors and each branch reaction, it is capable to catalyze the p-coumaric acid、erucic acid、caffeic acid and ferulic acid to generate their corresponding CoA ester.This study used the cDNA of Betula platyphylla as material, constructed plants over-expression vectors pROKII-4CL1、pROKII-4CL2、pROKII-4CL3and antisense expression vector pROKII-anti4CL1according to the4CL cDNA full-length gene obtained early days, and gained Bp14CL1-overexpressing positive plants using Agrobacterium-mediated genetic transformation. Both of the PCR detections using NPT II specific primers and Bp14CL1gene-specific primers generated specific bands. The results of qRT-PCR showed that the expression levels of Bp14CL1gene in transgenic lines was significantly higher than those in the control lines, demonstrated that the Bp14CL1gene has already been conformited in the Betula platyphylla genome and expressed at RNA level. Four shoots with Kanamycin resistance were obtained in this study, three of which were approved to be transgenic lines after detection.35transgenic plants were obtained in total and the positive efficiency of shoots with Kanamycin resistance was about75%. In addition, there was also a optimization of the genetic transformation program of Betula platyphylla. The best program is:pre-cultured for3days, co-cultured for3days, infected for5minutes. The optimal concentration of kanamycin is40mg/L for selection, and the optimal concentration of Carbenicillin is500mg/L for cleaning up the agrobacterium.