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原花青素是黑果枸杞中一类重要的植化产物,但其生物合成机制仍不清楚。本研究利用黑果枸杞EST数据库,克隆了无色花色素还原酶基因(LrLAR)和花青素还原酶基因(LrANR)。PCR结果表明,LrLAR和LrANR分别由333个氨基酸残基、338个氨基酸残基组成的蛋白编码。进化分析表明,相应蛋白LrLAR、LrANR分别聚类于LAR和ANR簇。实时定量PCR结果表明,从果实未成熟时期到变色期,LrLAR、LrANR的表达量均急剧上升,但在随后的发育时期中逐渐下降;与在果实中相比,在嫩叶、成熟叶、茎、根中,两基因的表达量极低。植化分析发现,果实中总原花青素含量高于嫩叶、成熟叶、茎、根中的含量,并且在果实成熟过程中呈现先增加后基本趋于稳定的趋势。本研究将为揭示黑果枸杞原花青素生物合成机制及促进其工程改良奠定基础。
Proanthocyanidins are one of the important phytochemicals in Lycium barbarum, but its biosynthesis mechanism remains unclear. In this study, the colorless anthocyanin reductase gene (LrLAR) and the anthocyanin reductase gene (LrANR) were cloned using the black fruit Lycium barbarum EST database. PCR results showed that LrLAR and LrANR were encoded by a protein consisting of 333 amino acid residues and 338 amino acid residues, respectively. Phylogenetic analysis showed that the corresponding proteins LrLAR and LrANR clustered in LAR and ANR clusters, respectively. The results of real-time quantitative PCR showed that the expression levels of LrLAR and LrANR increased sharply from the immature fruit stage to the discolouration stage, but decreased gradually in the subsequent developmental stages. Compared with the fruits, the young leaves, the mature leaves and the stems In roots, the expression of both genes is extremely low. Phytochemical analysis showed that the content of total proanthocyanidins in fruits was higher than that in young leaves, mature leaves, stems and roots, and tended to be stable after the first increase in fruit ripening. This study will lay the foundation for revealing the mechanism of biosynthesis of proanthocyanidins from Lycium barbarum and promoting its engineering improvement.