| Tea polyphenols are synthesized through phenylalanine pathway and flavonoids synthesispathway, and CHS is one of the most important enzyme proteins in the pathway. CHS catalyzes onecoumaric acid CoA and3Malonyl-CoA to condensate into a chalcone, which is a branch channelbetween the synthesis of lignin and flavonoids. Enzyme CHS is coded by CHS gene, and flavonoidcontent in plant is significantly related to CHS gene.This research begins from gDNA and cDNA sequences clone of CHS1, CHS2and CHS3fromCamellia sinensis, and gene structures are determined. Then Real Time PCR （RT PCR）is used todetermine the relative levels of expression of these three genes in vegetative organs and reproductiveorgans, respectively. According to the differences among three CHS genes’ expression, the key CHSgene in vegetative organs of tea plant is determined. Thirdly, we discussed the relationship ofpolyphenols level and key CHS gene’s relative expression level in leaves of new shoots in tea plants. Atlast,57sequences of key CHS are received using PCR method, and correlation analysis was used todiscuss the relationship between SNP sites and tea polyphenols content. The main results are:1) CHS1, CHS2and CHS3genes’ gDNA and cDNA sequences were obtained using PCR method, andgene structures were determined by sequence blast: there are2exons and1intron in both CHS1andCHS3, intron in both genes is located between the first and second sites in codon of60thCys. Intronin CHS1and CHS3is323bp and365bp, respectively. No insert sequence was found in gDNA ofCHS2, and ‘AG’missing was found in CHS2of most varieties.2) Amino acid sequences and protein structures were deduced from cDNA of CHS1, CHS2andCHS3using SOPMA, SinalP, ProtScale and other softwares. Results: Identity among the threeamino acid sequences are above92.6%, main second structure in protein are αhelix and coil, andonly protein CHS3has a signal polypeptide composed of27amino acids in N terminal. All of thethree CHS protein are hydrophilicity, and they all have six modify sites, including N-glycosylationsite, cAMP-and cGMP-dependent protein kinase phosphorylation site, Protein kinase Cphosphorylation site, Casein kinase II phosphorylation site, N-myristoylation site, Leucine zipperpattern, Chalcone and stilbene synthases active site.3) Real Time PCR results: CHS2is the key CHS gene in vegetative organs, while CHS1is the keyCHS gene in reproductive organs of Camellia sinensis. In vegetative organs, CHS expressed most insteam, then in leaves of new shoots, expressed least in mature leaves. In reproductive organs, CHSexpressed most in petals and stamens, expressed in very low levels in calyx, pollen and style. Thereis a synergistic increase or decrease relationship between tea polyphenol level and relativeexpression level of CHS2in leaves.4)57sequences of CHS2were obtained by sequencing PCR products directly, among which55SNPsites were found. And4SNP sites were found to be significantly related to tea polyphenol levels. |
