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园艺学报 ›› 2014, Vol. 41 ›› Issue (4): 701-712.

• 蔬菜 • 上一篇    下一篇

紫山药花青素调控基因DaF3H的克隆及表达分析

闫瑞霞,殷剑美*,韩晓勇,张培通   

  1. (江苏省农业科学院经济作物研究所,南京 210014)
  • 收稿日期:2013-11-08 出版日期:2014-04-25 发布日期:2014-04-25

Cloning and Expression Analysis of DaF3H Gene in Yam(Dioscorea alata)

YAN Rui-xia,YIN Jian-mei*,HAN Xiao-yong,and ZHANG Pei-tong   

  1. (Institute of Industrial Crops,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)
  • Received:2013-11-08 Online:2014-04-25 Published:2014-04-25

摘要: 以富含花青素的紫山药‘蓣引紫006’为材料,克隆得到花青素生物合成途径中关键酶基因DaF3H(KF561995),其序列全长为1 325 bp,最大开放阅读框为1 089 bp,编码362个氨基酸。DaF3H蛋白属于水溶性的胞质不稳定蛋白,无跨膜区和信号肽结构,为α–酮戊二酸和二价铁离子依赖型的加氧酶家族[2OG-Fe(Ⅱ)Oxygenase superfamily]。氨基酸序列与胡桃(ACR47976)、琴叶拟南芥(CAC26921)相似性最高,达到80%DaF3H在山药幼叶中表达量最高,在成熟叶片和茎中几乎不表达。茎叶和块茎中花青素积累量增速与DaF3H表达密切相关。运用接头PCR法对DaF3H的启动子序列进行了克隆,元件预测显示该序列中存在GT-1光调控元件以及生长素、金属离子、MYB-bZIP-MYC等元件的结合位点,叶片遮光处理试验表明,DaF3H受光调节。

关键词: 山药, DaF3H, 花青素, 启动子克隆, 表达分析

Abstract: DaF3H,a key gene of anthocyanin biosynthesis,has been isolated from‘Yzi 006’,a purple yam cultivar riching in anthocyanins. DaF3H(KF561995),full sequence of 1 325 bp,had a open reading frame of 1 089 bp encoding 362 amino acids. The DaF3H protein belonged to soluble protein without signal peptide and transmembrane domain,and localized in cytoplasm. The conserved domain search revealed DaF3H belonged to the 2OG-Fe(Ⅱ)oxygenase superfamily. Comparative and bioinformatic analysis showed DaF3H was highly homologous with F3Hs from other plants,such as Juglans regia and Arabidopsis lyrata ssp. petraea(80%). Expression analysis indicated DaF3H gene had the highest relative expression level in young leaves and less in functional leaves and stems. Anthocyanins accumulatedtrends and DaF3H gene expression level were closely related in leaves,stems and storage stems of yam. Furthermore,a 502 bp nucleotide promoter sequence of DaF3H has been isolated by adapter PCR method and the cis-regulating element analysis predicted there were many elements regulating the expression level of DaF3H in yam,such as auxin,metal ion,MYB-bZIP-MYC complex and GT-1binding sites for light regulation which was proved by the experiment on leaves treated in dark.

Key words: Dioscorea alata, DaF3H, anthocyanins, promoter cloning, expression analysis

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