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木脂素是重要的植物防御物质之一,具有优良的生物学活性。为研究传统抗肿瘤良药野生金荞麦的药用次生代谢,用RACE与文库结合的方法,从野生金荞麦高黄酮含量愈伤系cDNA文库中快速克隆了一个类异黄酮还原酶(isoflavone reductase-like,IRL)基因FcIRL的全长cDNA(accession no.EU116032)。FcIRL cDNA全长1 217 bp,含有1个942 bp的开放阅读框(open reading frame,ORF),编码313个氨基酸,其5′UTR区含有2个终止子TAG,3′UTR区含有推测的加尾信号ATAAA和polyA尾,对应基因组序列不含内含子。FcIRL蛋白N端含有1个N-乙酰化作用位点(M1-K5)和1个保守的NADPH结合位点(G10-G-T-G13-Y-I-G16),具有1个保守的NmrA结构域(V6-N244)和多个重要的磷酸化位点,以及1个保守的N-糖基化作用位点(N214)。序列同源性比较、系统发生分析以及蛋白质高级结构预测分析均表明,FcIRL属于松脂醇-落叶松脂醇还原酶(pinoresinol-lariciresinol reductase,PLR)类,推测具有PLR的生物活性,是8-8′连接木脂素(8-8′-linked lignans)合成途径中的关键酶,催化松脂醇和落叶松脂醇,还原生成开环异落叶松脂(secoisolariciresinol),参与金荞麦药用次生代谢与植物的防御。
Lignans are one of the important plant defense materials and have excellent biological activity. In order to study the medicinal secondary metabolism of the traditional anti-tumor medicine, wild gold buckwheat, an isoflavone reductase (isoflavone reductase-) was rapidly cloned from the callus cDNA library of wild golden buckwheat and high flavone content using a combination of RACE and library methods. Like, IRL) Full-length cDNA of the gene FcIRL (accession no. EU116032). The FcIRL cDNA is 1 217 bp in length and contains a 942 bp open reading frame (ORF) encoding 313 amino acids. The 5′ UTR region contains 2 terminator TAGs. The 3′ UTR region contains putative plus. Tail signal ATAAA and polyA tail, the corresponding genome sequence does not contain introns. The N-terminus of FcIRL protein contains an N-acetylation site (M1-K5) and a conserved NADPH binding site (G10-GT-G13-YI-G16) with a conserved NmrA domain (V6 -N244) and several important phosphorylation sites, as well as a conserved N-glycosylation site (N214). Sequence homology comparisons, phylogenetic analysis, and protein structural predictions all indicate that FcIRL belongs to the pinoresinol-lariciresinol reductase (PLR) class and presumably has the biological activity of PLR, which is 8-8′. Ligation of key enzymes in the synthesis pathway of lignans (8-8′-linked lignans), which catalyzes pinoresinol and larchol esters to reduce the formation of secoisolariciresinol, which is involved in medicinal secondary metabolism and defense of plants. .