Ma Jing, Wang Bin, Dai Yin, Sui Shun-Zhao, Li Ming-Yang
College of Horticulture and Landscape, Southwest University, Key Laboratory of Horticulture Science for Southern Mountainous Regions of Ministry of Education, Chongqing Engineering Research Center for Floriculture, Chongqing 400716, China.
Yao Xue Xue Bao. 2012 Jul;47(7):953-61.
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 degrees C and induced by IPTG at final concentration of 1.0 mmol x 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.
通过简并PCR和cDNA末端快速扩增（RACE）技术，从金荞麦（Fagopyrum dibotrys (D.Don) Hara）中克隆了儿茶素生物合成途径的一个重要功能基因——无色花青素还原酶（LAR）基因。金荞麦FdLAR的全长cDNA为1581 bp（GenBank登录号：JN793953），包含一个1176 bp的开放阅读框，编码一个391个氨基酸的蛋白质，其3'非翻译区有一个明显的多聚腺苷酸化信号。将含有FdLAR完整开放阅读框的重组质粒转化到大肠杆菌BL21(DE3)中。在16℃条件下，用终浓度为1.0 mmol·L⁻¹的异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达分子量为66 kD的目标融合肽。生物信息学分析表明，FdLAR的氨基酸序列与其他在N端具有NADB-Rossmann保守结构域的LAR具有高度同源性。采用实时定量PCR检测FdLAR基因在不同发育时期的表达水平。对金荞麦根状茎中黄酮类化合物含量的测定表明，在营养生长和生殖生长阶段，FdLAR基因表达与黄酮类化合物积累之间的关系呈现不同趋势，这表明FdLAR基因可能参与了金荞麦黄酮类代谢途径。
