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无色花色素还原酶(leucoanthocyantin reducase,LAR)基因是植物类黄酮代谢途径中催化缩合单宁合成的一个关键结构基因,本研究通过简并PCR结合RACE的方法,获得了1个金荞麦(Fagopyrum dibotrys(D.Don)Hara)无色花色素还原酶基因FdLAR(GenBank accession:JN793953),序列全长1 581 bp,其中开放阅读框长1 176 bp,编码391个氨基酸的蛋白质,在N端存在1个保守结构域,属于RED蛋白家族。将该基因重组到表达载体pET-32a(+)中进行原核表达,经IPTG诱导、SDS-PAGE检测,结果表明金荞麦无色花色素还原酶基因能在大肠杆菌BL21(DE3)中表达,电泳检测到1条大约66 kD的外源蛋白,与预测的融合蛋白分子量相符。利用实时荧光定量PCR技术检测FdLAR基因在金荞麦根茎中不同生长发育时期的表达情况,同时测定相应根茎中类黄酮的含量,结果表明FdLAR基因的表达量与类黄酮积累之间的关系在营养生长和生殖生长阶段呈现出不同的变化趋势,推测该基因可能在金荞麦类黄酮次生代谢产物积累中起作用。
The gene of leucoanthocyantin reductase (LAR) is one of the key structural genes that catalyze the synthesis of tannin in plant flavonoid metabolism pathway. In this study, we obtained 1 Fagopyrum (Fagopyrum) (GenBank accession: JN793953) with a full-length of 1 581 bp, including an open reading frame (1 176 bp) encoding a 391 amino acid protein at the N-terminus One conserved domain, belonging to the RED protein family. The gene was recombined into the expression vector pET-32a (+) for prokaryotic expression, induced by IPTG and detected by SDS-PAGE. The results showed that the gene was expressed in E. coli BL21 (DE3) A foreign protein of about 66 kD was detected, which was consistent with the molecular weight of the predicted fusion protein. FdLAR gene was detected by real-time fluorescence quantitative PCR at the different growth and development stages of F buckle root rhizomes, and the content of flavonoids in the corresponding rhizomes was determined. The results showed that the relationship between FdLAR gene expression and flavonoid accumulation in vegetative growth And reproductive growth stage showed different trends, suggesting that the gene may play a role in the accumulation of secondary metabolites of buckwheat flavonoids.