摘要：

花色苷是植物次生代谢过程中产生的黄酮类物质,在花色苷合成途径中,花色苷合成酶催化无色花色苷转化成有色的花色苷,在植物器官着色过程中起重要作用。为了解彩色马铃薯花色苷合成途径中关键酶基因ANS的功能,以红皮红肉的彩色马铃薯品种红美为试验材料,利用RT-PCR技术克隆花色苷合成酶基因,通过生物信息学分析其特性,并将其过表达到拟南芥中进行功能验证。结果显示,该基因cDNA序列长为1 406 bp,包含1 368 bp完整的开放阅读框,编码 455个氨基酸残基。通过生物信息学分析克隆到的基因,并将该基因命名为StANS1a,GenBank登录号为ON512347,其氨基酸序列具有保守的结构域DIOX_N和2OG_FeⅡ_Oxy,分别位于氨基酸序列的52~166位和 215~312位。在拟南芥中过表达StANS1a,通过半定量RT-PCR检测其转录水平,转基因株系中都有StANS1a基因的表达,且表达量较非转基因拟南芥高;通过检测转基因植株中花色苷的含量,发现转基因植株花色苷含量比非转基因植株高2.17%~54.61%,表明StANS1a参与了花色苷的生物合成。同时也证明彩色马铃薯StANS1a基因在花色苷代谢途径中起着重要的作用。

关键词: 彩色马铃薯, 花色苷合成酶基因, 基因克隆, 表达分析, 花色苷

Abstract:

Anthocyanins are a kind of important flavonoids produced during the secondary metabolism of plants.Anthocyanidin synthase(ANS)catalyzes the conversion of leucoanthocyanidins to anthocyanidins in the anthocyanin synthesis pathway.Previous studies have shown that the anthocyanidin synthase gene plays an important role during the coloring process of plant organs.To investigate whether the gene can promote anthocyanin synthesis in potato tubers,using the red-skinned and red-fleshed colored potato variety Hongmei as the material,anthocyanidin synthasegene was isolated from tubers of color potato by RT-PCR, the characterization analyzed by bioinformatics and function validated by overexpression into Arabidopsis thaliana.The results showed that sequence 1 406 bp cDNA of the gene was amplified from the potato,including 1 368 bp open reading frame(ORF).The ORF encoded a deduced protein of 455 amino acids.The gene was named as StANS1a by bioinformatics analysis.GenBank accession number is ON512347.The amino acid sequence analysis revealed that the deduced protein contained a DIOX_N'domain located at 52—166 resides and a 2OG_FeⅡ_Oxy domain located at 215—312 resides.Arabidopsis thaliana plants was transformed with constructs overexpression vector StANS1a.The transcriptional expression of StANS1a in transgenic lines were tested by RT-PCR.The results showed that in the same population,the transcriptional expression of StANS1a showed an increase in different transgenic lines compared with the wild-type(Wt).The anthocyanin content was further analyzed in transgenic lines.Compared to the Wt,the anthocyanin contents of the seven lines(T₁—T₇)were increased from 2.17% to 54.61%. These results strongly demonstrated that overexpression of the StANS1a gene could promote anthocyanin synthesis in transgenic plants.The colored potato StANS1a gene plays an important role in the anthocyanin metabolic pathway.

Key words: Color potato, Anthocyanidin synthase gene, Gene clone, Expression analysis, Anthocyanins