金荞麦无色花色素还原酶基因FdLAR的克隆和表达分析

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。

Cloning and expression analysis of leucoanthocyanidin reductase gene in Fagopyrum dibotrys

The leucoanthocyantin reducase(LAR) gene,an important functional gene of catechins biosynthesis pathway,was cloned from Fagopyrum dibotrys(D.Don) Hara by degenerate PCR and rapid amplification of cDNA ends(RACE).The full-length cDNA of FdLAR is 1 581 bp(GenBank accession: JN793953),containing a 1 176 bp ORF encoding a 391 amino acids protein,and its 3’-untranslated region has an obvious polyadenylation signal.The recombinant plasmid containing FdLAR completed ORF was transformed into E.coli BL21(DE3).The target fusion peptide with molecular weight of 66 kD was expressed under the condition of 16 ℃ and induced by IPTG at final concentration of 1.0 mmol.L 1.Bioinformation analysis indicated that the amino acid sequence of FdLAR showed great homology to other LAR with the NADB-Rossmann conversed domain in the N-terminus.Real-time quantitative PCR was used to detect the expression levels of FdLAR gene during different development periods.The determination of flavonoids contents in appropriate rhizomes showed that the relationship between FdLAR gene expression and the accumulation of flavonoids displayed different trends during vegetative growth and reproductive growth stages,suggesting that the FdLAR gene may be involved in the pathway of flavonoid metabolisms in Fagopyrum dibotrys.