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

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

【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。

【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.