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期 刊 ：植物遗传资源学报 2014年 15卷 5期

关键词：关键词烟草；肉桂酸-4-羟化酶(C4H)；4-香豆酸-辅酶A(4CL)；基因分离；表达分析；

Keywords:Tobacco, Phenylpropanoid metabolic pathway, Cinnamic acid -4- hydroxylase (C4H), Coumaric acid -4- ligase (4CL), Gene isolation, Expression anlysis,

摘 要 ：肉桂酸-4-羟化酶（C4H）、4-香豆酸-辅酶A（4CL）是烟草苯丙烷代谢途径的关键酶，其多酚类产物与烟草品质密切相关。本研究以酚类物质含量合成差异较大的两烤烟品种红花大金元（HD）和K326为实验材料，利用同源克隆技术获得这两种烟草Ntc4h和Nt4cl基因的cDNA序列并进行表达特性分析。结果表明，在两品种中Ntc4h和Nt4cl各有两个同源基因，Ntc4h1、Ntc4h2、Nt4cl1和Nt4cl2的ORF长度分别为1518 bp、1518 bp、1644 bp和1629 bp。Nt4cl1、Ntc4h1和Ntc4h2在编码序列上存在品种间差异。实时荧光定量PCR分析结果表明，该两种酶的基因在烟草中具有明显的时空表达特异性，两种酶基因在根、茎、叶、花和萼片中都有表达，在茎的木质部和韧皮部中的表达量均显著高于其他组织；在圆顶期和适熟期表达水平较高，在适熟期达最高；在两品种中的表达模式存在差异。

Abstract:Cinnamic acid -4- hydroxylase (C4H) and ρ-coumaric acid -4- ligase (4CL) are the key enzymes in phenylpropanoid metabolic pathway, whose polyphenol products is corresponding to the quality of tobaccos. Here, cDNAs of these two enzymes were obtained using homology-based cloning from variety honghua dajinyuan (HD) and variety K326, whose quantity of polyphenol products differed significantly and their expression profiles were analyzed by Real time qRT-PCR. The results showed that Ntc4h and Nt4cl both have two homologous genes. The ORF length of Ntc4h1, Ntc4h2, Nt4cl1 and Nt4cl2 was 1518 bp, 1518 bp, 1644 bp and 1629 bp, respectively. According to the analysis, Ntc4h1, Nt4cl1 and Ntc4h2 have shown differences in these two varieties on coding sequence. Real time qRT-PCR analysis showed that all the genes expressed in a spatial and temporal manner and the expression profiles differed in two varieties. The genes all expressed in root, stem, leaf, flower and sepal with the expression of xylem and phloem were higher than others. The expression in fast growing stage and technical maturity stage was higher than the others with the highest in technical maturity stage.

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