In order to explore the molecular mechanism of Chinese fir wood formation, total RNA was extracted from Chinese fir new leaves and cDNA was synthesized. A new ClCesA was cloned using RT-PCR. The length of open reading frame of ClCesA is 2955 bp, with 984 amino acids. Based on TMHMM Server v. 2.0 and MEGA5.1 software, ClCesA proteins consist of 8 across-membrane structures, with average 18 amino acid residues and zinc finger in N end. Also, a conservative DDDQXXLRW structure domain was found in ClCesA protein. Phylogenetic tree analysis showed that ClCesA may be associated with secondary cell wall formation. The flu-orescent quantitative PCR analysis showed that ClCesA can be expressed in organs of root, stem and leaf in Chinese fir, with the highest expression in the root and the least in leaf. Lastly, a plant overexpression vector pGWB506-35s-ClCesA-GFP was successful-ly constructed from pGWB506 vector which contains GFP tag and hygromycin resistance gene by Gateway technology.%为探索杉木木材形成的分子机制,以杉木嫩叶总RNA反转录的cDNA为模板,运用RT-PCR技术克隆出一个新杉木纤维素合酶基因ClCesA.该基因的开放阅读框(ORF)为2955 bp,共编码984个氨基酸.通过TMHMM Server v.2.0和MEGA 5.1软件预测ClCesA蛋白的跨膜结构和功能,发现该蛋白N端含有锌指结构,共有8个跨膜结构,平均跨膜区域18个氨基酸残基,并具有保守的DDDQXXLRW结构域.系统进化树分析结果表明,ClCesA可能与细胞次生壁形成有关.荧光定量PCR分析表明,ClCseA基因在杉木的不同器官(根、茎、叶)中均有表达,但在杉木根中的表达含量最高,茎中次之,叶中最低.随后,为后续该基因功能验证提供基础,将ClCesA克隆到含有GFP标签和潮霉素抗性的pGWB506载体,构建植物过表达载体pGWB506-35S-ClCesA-GFP.
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