苯丙氨酸与UV-C对苦荞芦丁含量影响及相关基因表达分析

 【目的】克隆与苦荞芦丁生物合成相关基因序列，探讨外源前体物质及UV-C辐射条件下芦丁含量及基因表达水平的影响，为分子选育高芦丁含量荞麦品种奠定基础。【方法】基于简并引物结合3′RACE的方法，克隆苦荞芦丁合成途径相关基因；高效液相色谱（HPLC）测定持续添加外源苯丙氨酸（L-phenylalanine，Phe）前体（1、2、4 mg?L-1）1—5 d和辐射时间（1、3、5、7 h）UV-C处理条件下叶片芦丁含量的变化，qRT-PCR分析相关基因表达水平。【结果】Phe（4 mg?L-1）处理1 d，芦丁含量（26.15 mg?gFW-1）为未处理对照（12.55 mg?gFW-1）的2倍；UV-C辐射3 h，芦丁含量（17.36 mg?gFW-1）明显高于对照；克隆到3个基因FtCHS，FtF3H和FtFLS-like，qRT-PCR分析表明Phe（1 mg?L-1）处理4 d，FtCHS相对表达量为对照的4.84倍，Phe（4 mg?L-1）处理5 d，FtF3H相对表达量为对照的1.06倍，Phe（2 mg?L-1）处理5 d，FtFLS-like相对表达量为对照的3.90倍；UV-C辐射1—3 h，FtCHS、FtF3H和FtFLS-like相对表达量分别高于对照。而FtFLS-like基因，仅在UV-C辐射1 h，相对表达量升高为对照的127.71倍。【结论】添加不同浓度Phe前体及不同时间UV-C辐射，芦丁含量变化及与芦丁合成相关基因表达量存在差异，为进一步探讨芦丁生物合成途径及影响因素奠定基础。

Effect of Exogenous L-Phentermine and UV-C on the Accumulation of Rutin Compounds and the Expression of Rutin Biosynthesis Genes in Fagopyrum tartaricum

【Objective】 The objective of this study is to investigate the content of rutin and the expression of the related genes in response to L-phentermine (Phe) and UV-C treatment, and to provide molecular information for high rutin content buckwheat breeding. 【Method】 Rutin biosynthesis-related genes were cloned from the leaves of F. tataricum by 3′RACE. Rutin content in leaves under Phe treatment (1, 2 and 4 mg?L-1, 1 to 5 d) was compared with that of untreated buckwheat by high performance liquid chromatography (HPLC). The gene expression level was analyzed by qRT-PCR. 【Result】 Three partial CDS of rutin biosynthesis-related genes were cloned. They were chalcone synthase (FtCHS), flavonol synthase-like (FtFLS-like) and flavone 3-hydroxylase (FtF3H). Seedlings were cultured in solution containing Phe (4 mg×L-1) for 1 d, rutin content was two times more than the control (12.55 mg×gFW-1), reached 26.15 mg×gFW-1. The higher rutin content (17.36 mg×gFW-1) was found in leaves at 3 h under UV-C treatment. Differential expression patterns of FtCHS, FtFLS-like and FtF3H were observed in different Phe concentration treatments in leaves of F. tartaricum. Seedlings were cultured in solution containing Phe (1 mg×L-1) for 4 d, FtCHS expression  level was 4.84 fold higher than the control. The higher expression levels of FtF3H and FtFLS-like were observed after 4 and 2 mg×L-1 Phe treatment for 5 d, they were 1.06 and 3.90 fold higher than the control, respectively. UV-C radiation for 1-3 h, the expression      of FtCHS, FtFLS-like and FtF3H was higher than control. In particular, the expression level of FtFLS-like at 1 h under UV-C radiation was 127.71 fold higher than the control. 【Conclusion】 Rutin content could be enhanced to a great extent by Phe treatment and UV-C radiation, and gene expression patterns of rutin biosysthesis-related genes were regulated by UV-C radiation. It can be used to analyze the pathway of rutin biosynthesis. Further study should be focused on the influential factors of rutin metabolic process.