茶树CsCDF1基因克隆及表达分析

采用SMART-RACE技术克隆了茶树Dof（DNA binding with one finger）基因CsCDF1的全长cDNA序列,并利用在线生物信息学软件对其进行了分析。采用实时荧光定量PCR分析该Dof基因在茶树不同组织间的表达差异和昼夜表达规律,及其在氮饥饿处理2周后对不同浓度氮素诱导的响应。该cDNA序列全长1β606βbp,包含1个编码464个氨基酸的完整开放阅读框,含有高度保守的DOF结构域,推导的蛋白分子量为50.8βkDa,理论等电点（PI）为5.52;其编码的蛋白序列与茸毛烟草、马铃薯和美花烟草的CDF蛋白（Cycling dof factor）相似性分别为69%、67%、68%。系统发育分析结果表明,该基因编码的氨基酸序列与拟南芥CDF蛋白聚为一类,因此将其命名为CsCDF1;CsCDF1在3个不同茶树品种根系中的表达量均高于一芽二叶和成熟叶;在一天中,该基因的表达呈现出昼夜节律变化;在氮饥饿后重新供氮,不同组织中该基因对不同浓度氮素的响应均为上调。

Cloning and Expression Analysis of CsCDF1 (Cycling Dof Factor 1) Gene in Tea Plant (Camellia sinensis)

The full-length cDNA of the firstDofgene (CsCDF1)was cloned from tea plant [Camellia sinensis(L.) O. Kuntze] by SMART-RACE cloning technology, and the bioinformatic analysis of the cloned gene were conducted by using online service.The expression profile of this gene in various tissues and with diurnal rhythm as well as in response to different dosage nitrogen treatment were investigated by using real-time fluorescent quantitative RT- PCR. The obtained cDNA sequence was 1 606 bp and contained a complete open reading frame encoding 464 amino acid residues with highly conserved DOF domain. The molecular weight and theoretical isoelectric point (PI) is 50.8 kDa and 5.52 respectively. The analysis by utilizing the BLAST software showed that the derived protein sequences shared separately 69%, 67% and 68% similarity with CDF (Cycling dof factor) proteins inNicotianato-mentosiformis, Solanum tuberosum andNicotiana sylvestris. Phylogenetic analysis showed that the protein encoded by this gene was clustered into the same clade withArabidopsis thaliana CDF, so it was named asCsCDF1. The expression ofCsCDF1 dominated in roots rather than in one bud and two leaves or mature leaves in three phenotypes. During one day,CsCDF1gene changed with the diurnal rhythm, and resupplying nitrogen after starvation for 2 weeks, the expressionin response to different concentration of nitrogen was all raised in different tissues.