苋菜amaARF6基因克隆及其外源激素处理下在幼苗中响应分析

Cloning of amaARF6 and its response to exogenous hormones treatment in amaranth seedlings

以‘大红’苋菜(Amaranthus tricolor L.‘Dahong’)为材料,根据实验室构建的苋菜转录组数据库(SRA:SSR924089~SSR924092),采用RT-PCR结合RACE技术克隆获得1条生长素响应因子ARF6基因c DNA全长序列,命名为amaARF6(登录号为MG581459)。苋菜amaARF6基因c DNA全长3 758 bp,含一个2 697 bp开放阅读框,编码898个氨基酸。生物信息学分析表明,amaARF6包括生长素响应因子结构域和生长素功能位点,有ARF基因家族特有序列特征;系统进化树分析发现,amaARF6蛋白与同为石竹目甜菜Bv ARF6蛋白进化距离最近,相似度高达100%。荧光定量PCR分析表明,苋菜幼苗在不同浓度2,4-D、IAA、IBA等生长素及不同浓度GA3和PP333处理下,amaARF6基因均受调控,且在2,4-D、IAA、IBA等生长素处理后表达上调,经GA3和PP333处理后表达下调。研究还发现,苋菜中甜菜红素累积受GA3和2,4-D抑制,PP333促进甜菜红素合成,amaARF6基因表达量在GA3和PP333处理下呈相反变化趋势。研究结果表明,amaARF6基因可能具备ARF家族正调控因子表达特征和功能,可能参与调控苋菜幼苗生长和甜菜红素合成。研究结果为揭示amaARF6基因在苋菜幼苗生长中作用奠定基础,为苋菜育种提供线索。

According to the database of amaranth transcriptome （SRA： SSR924089-SSR924092） constructed by the laboratory, one full-length amaARF6 cDNA sequence was cloned from Amaranthus tricolor L. ＇Dahong＇ by RT-PCR and RACE technologies and named amaARF6 （Accession number is MG581459）. The full-length amaARF6 cDNA sequence was 3 758 bp and contained a 2 697 bp open reading frame encoding 898 amino acids. Bioinformatics analysis showed that amaARF6 included the auxin response factor domain and the auxin functional site with sequences specific to ARF gene family. The phylogenetic tree analysis showed that the closest evolutionary distance between amaARF6 protein and BvARF6 protein was about 100%. Quantitative real-time PCR analysis showed that amaARF6 gene was regulated by these exogenous hormones under different concentrations of 2, 4-D, IAA, IBA and other auxins and different concentrations of GA3 and PP333, 2, 4-D, IAA, IBA and other auxin upregulated, down-regulated by GA3 and PP333 treatment. The study also found that the accumulation of betacyanin in amaranth was inhibited by GA3 and 2, 4-D while PP333 promoted the synthesis of betacyanin, and the expression of amaARF6 gene showed the opposite trend under GA3 and PP333 treatments. The results showed that amaARF6 gene might possess the expression characteristics and functions of positive regulators of ARF family, and might be involved in the regulation of the growth of amaranth seedlings and the synthesis of betacyanin. This study might lay a foundation for revealing the role of amaARF6 gene in the growth of amaranth seedlings and provide clues for the breeding of betacyanin-rich amaranth.