| The Vine Tea Ampelopsis grossedentata W.T.Wang, is a Vitaceae Ampelopsis vine. Its stems and leaves are rich in flavonoid compounds such as dihydromyricetin (DMY) and can be processed into health tea with many medicinal efficacies such as detoxification, anti-inflammatory, antioxidant, hepatoprotective, and immunity enhancement.In this study, five genes related to flavonoid biosynthesis were cloned from ampelopsis, and reference genes suitable for real-time fluorescence quantitative PCR (qRT-PCR) technique were screened out. Then the expression levels of flavonoids synthesis-related genes in different tissues and at different developmental stages and their correlations with the content of DMY were analyzed in vine leaves, in order to provide basic information to clarify the molecular mechanism of high content of DMY of ampelopsis. The main results are as follows:1.The expressions of six housekeeping genes, including Actin,18s-rRNA, GAPDH, α-tubulin, β-tubulin, and UBQ, were detected in ampelopsis and the data were analyzed by using geNorm, NormFinder, and BestKeeper softwares. The results showed that the stability of Actin,18s-rRNA, and GAPDH was good when the gene expression was compared in different organs and tissues; the stability of 18s-rRNA, GAPDH, and a-Tubulin was good when the gene expression was compared at different development stages of the leaves. After comprehensive consideration, 18s-rRNA was selected as the reference gene in this study.2.By homology cloning technology, five flavonoid biosynthesis-related genes were cloned from ampelopsis leaves, namely, AgPAL, AgC4H, Ag4CL, AgCHS, and AgCHI, with an open reading frame length of 2133bp,1518bp,1713bp,1182bp, and 717bp, respectively. BLAST analysis of NCBI database showed that the polypeptide sequences derived from these five genes were highly homologous with those of other species such as grape, birch, honeysuckle, and orange (77%-96%).3.qRT-RCR results showed that the expression levels of AgPAL, AgC4H, Ag4CL, and AgF3H were decreased with the increase of leaf maturity in the first three leaves at different developmental stages, those of the fourth leaf. however, were increased; the expression levels of AgF3’H in the first and second leaves showed a decreasing trend, while those in the third and fourth leaves were in increasing state; the highest expression levels of AgCHS and AgF3’5 H were found in the first leaf, with no significant variance in the expression levels from the second to the fourth leaf; no significant changes were found in the expression levels of AgCHI and AgDFR from the first to the fourth leaf. For different organs, the highest expression levels of AgPAL and AgDFR were found in radicles, followed by leaves and young stems; Ag4CL, AgCHS, and AgF3’5’ H expressed only in leaves, with almost no expression in radicles and young stems; the expressions of AgC4H, AgCHI, AgF3H, and AgF3’H reached their highest levels in leaves, followed by radicles, with the lowest levels in young stems.4.The contents of DMY in ampelopsis in different organs at different stages were measured by HPLC. The results showed that DMY contents decreased with the increase of leaf maturity at different developmental stages, i.e., the first leaf> the second leaf> the third leaf> the fourth leaf. For the different organs and tissues, the highest content of DMY was found in leaves, followed by young stems, and basically no DMY was detected in radicles.5.Correlation between DMY contents and the expression levels of flavonoid biosynthesis-related genes in different tissues and organs at different developmental stages of ampelopsis leaves was analyzed. Results showed that for the leaves at different growth stages, the higher expressions of AgPAL, AgC4H, Ag4CL, AgCHS, AgF3H, and AgF3’5 H went with the higher DMY contents, with a greater relationship with DMY; for different organs, DMY contents decreased with the increasing expressions of AgPAL and AgDFR, and therefore the expressions of AgDFR and AgPAL could negatively regulate the DMY in different organs. |
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