Cloning And Expression And Functional Verification Of Phenylpropane Pathway Gene In Vitis Amurensis

The peel of Vitis amurensis is rich in many natural anthocyanins.This matter not only has a deep development potential in different fields of life,but can also make the plants present a tempting color to protect against harmful ultraviolet rays.Therefore,it is of great significance to explore the biosynthesis of Vamurensis anthocyanin.In this experiment,the peel of Vamurensis in different development phases was taken as the test material to investigate PAL,C4H and 4CL genes that are involved in the first three catalytic steps of phenylpropane metabolism that controls the biosynthesis of anthocyanin.Rapid amplification of cDNA ends(RACE)was adopted to obtain the full-length cDNA sequence of PAL,C4H and 4CL genes in V.amurensis,which were analzyed using bioinformatics.The expression levels of these three genes in the peel of Vamurensis in different development phases were analyzed using real-time fluorogenic quantitative polymerase chain reaction(PCR)technique.The target genes were expressed in the escherichia coli and the expression process was optimized and induced.Vamurensis C4H was transferred into the arabidopsis thaliana,which was taken as the acceptor material.The main research results are as follows:With the RACE method,the full-length cDNA sequence of PAL,C4H and 4CL genes was cloned from the peel of Vamurensis.The analysis results revealed that the full-length cDNA of WaPAL,PAL gene of Vamurensis was 2 130 bp and open reading frame(ORF)was 1 926 bp.A total of 641 amino acids were coded and the molecular weight was 61.06 kDa.The full-length cDNA of VaC4H,C4H gene of Vamurensis was 1 735 bp and ORF was 1 518 bp.A total of 505 amino acids were coded and the molecular weight was 57.70 kDa.The full-length cDNA of Va4CL,4CL gene of V.amurensis was 1 772 bp and ORF was 1 635 bp.A total of 544 anino acids were coded and the molecular weight was 61.07 kDa.The rules in the expression of PAL C4H and 4CL genes in the peel was explored in different development phases of V.amurensis and the results showed that the expression level of VaPAL was successively declined from the phase before coloring to the full ripe phase and was barely expressed in the phase at 50%coloring and 100%coloring.The expression abundance cof JVaC411 in differenl coloring phases of the peel of Vamurensis was high and its expression level was gradually increased during the whole growth process.The expression abundance of Va4CL reached the peak when the coloring of the peel of Vamurensis was 100%.Before this stage,its expression presented an increasing trend but declined during the maturing phase.Its relative expression was the lowest among the three genes.The recombinant plasmid pET28a-VaPAL,pET28a-VaC4Hand pET28a-Va4CL were transferred to competent cell BL21.IPTG was added in to induce the expression of thallus.In this research,the concentration of Isopropyl ?-D-1-thiogalactopyrano side(IPTG)was screened at 28? and the optimal induced concentration of PAL,C4H and 4CL protein was 1.0 mmol L-1,1.0 mmol L-1,1.2 mmol L-1 and the molecular weight was 61 kDa,57 kDa and 61 kDa.The Arabidopsis thaliana plant with transgenosis of VaC4H grew more rapidly than the wild type,The number of leaves was larger than that of the controls,the middle leave and the petiole were both longer than that of the controls.The color of the stem and the leaves was evidently changed,with a deep color of vinicolor.