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园艺学报 ›› 2015, Vol. 42 ›› Issue (4): 643-654.doi: 10.16420/j.issn.0513-353x.2014-0822

• 果树 • 上一篇    下一篇

银杏类黄酮3’ 羟化酶基因的克隆与表达分析

李琳玲,程 华,陈小玲,程水源   

  1. 1经济林木种质改良与资源综合利用湖北省重点实验室,湖北黄冈 438000;2武汉轻工大学生物与制药工程学院,武汉 430023;3黄冈师范学院生命科学学院,湖北黄冈438000
  • 出版日期:2015-04-25 发布日期:2015-04-25
  • 基金资助:
    国家自然科学基金项目(31400556,31270717);湖北省自然科学基金项目(2011CDA117,2013CFB471)

Molecular Cloning,Characterization and Expression of Flavonoid 3’ Hydroxylase-like Protein Gene from Ginkgo biloba

LI Lin-ling1,3,*,CHENG Hua1,3,*,CHEN Xiao-ling1,3,and CHENG Shui-yuan1,2,**   

  1. 1Economic Forest Germplasm Improvement and Comprehensive Utilization of Resources of Hubei Key Laboratories Huanggang,Hubei 438000,China;2Wuhan Polytechnic University,School of Biology and Pharmaceutical Engineering,Wuhan 430023,China;3College of Life Science,Huanggang Normal University,Huanggang,Hubei 438000,China
  • Online:2015-04-25 Published:2015-04-25

摘要: 利用简并PCR和RACE技术从银杏叶片中分离到黄酮3’羟化酶基因GbF3’H的cDNA全长序列(GenBank No.:KP056747)。其全长为2 144 bp,含有一个1 671 bp的开放式阅读框(ORF),编码556个氨基酸序列,预测氨基酸大小为63.47 kD,等电点为7.71。蛋白质同源序列分析表明,GbF3’H与其他物种F3’H同源性较低,而与菊苣(Cichorium intybus)同源性最高,为56.3%。同源建模分析显示GbF3’H与P450家族蛋白的三维结构及活性位点高度相似,最终定位于微粒体膜上。进化树分析结果显示GbF3’H与其他植物分化较早,序列差异较大。组织表达分析显示,GbF3’H在银杏不同组织中都有表达,其中雄蕊表达水平最高,其次为成熟叶。诱导表达分析显示,GbF3’H转录水平受UV-B、6-BA、SA、ABA和IAA影响诱导上调,而伤害处理对其表达量无明显影响。GbF3’H诱导表达模式与调控银杏黄酮含量变化呈正相关,其上游调控序列存在与黄酮调控相关的顺式元件,意味着GbF3’H可能参与了银杏黄酮类物质的合成代谢,并与黄酮类物质向花青素合成分流有关。

关键词: 银杏, 类黄酮3’羟化酶, 黄酮, 表达调控

Abstract: The full-length cDNA sequences of F3’H gene(designated as GbF3’H)were isolated from Ginkgo biloba for the first time,GenBank No. KP056747. The full-length cDNA of GbF3’H is 2 144 bp long and contains a 1 671 bp open reading frame(ORF)encoding a 556 amino acid protein,with a predicted molecular weight of about 63.47 kD and an isoelectric point of 7.71. Phylogenetic tree analysis revealed that GbF3’H shared the same ancestor with other F3’H,but the divergence time is early. Bioinformatics analysis show that GbF3’H have a signal recognition peptide and belong to a microsomal cytochrome P450-dependent monooxygenases multigene families. The expression analysis by RT-PCR showed that GbF3’H had tissue specific manner in G. biloba,and the highest expression in stamens,thenext in mature leaves and gynoecium which were great difference with other flavones biosynthetic pathway gene. GbF3’H transcription level was also found to be significant up-regulated by 6-BA,SA,ABA,IAA,but wound treatment had no significant change. It’s also found that the GbF3’H promoter sequence contain the regulation cis-acting element assist with flavonoids biosynthesis. The result suggested that the expression model of GbF3’H gene has close relation with the formation of flavonoids and anthocyanin biosynthesis,which indicated that the F3’H regulate the flavonoids transfering to anthocyanin in G. biloba.

Key words: Ginkgo biloba, flavonoid 3’-hydroxylase, flavonoids, transcript regulation

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