生长素响应因子(ARFs)是能够与生长素原初反应基因启动子区的生长素响应基元(TGTCTC)特异结合的一类转录因子,调控生长素应答基因的表达.采用SMART-RACE-PCR技术,获得茶树生长素响应因子基因CsARF1的全长cDNA序列(GenBank登录号为JX307853),并进行了生物信息学分析和表达分析.CsARF1基因cDNA全长3222 bp,包含2463 bp的开放阅读框(ORF),编码820个氨基酸残基.编码蛋白的分子量为49.35 kD,具有保守的N端DNA结合域 B3和 C 端二聚化结构域 IAA_ARF,中间区域富含谷氨酸、丝氨酸和亮氨酸,是一个定位于细胞质的具有激活转录功能的可溶性蛋白.相似性及系统进化分析表明,该基因编码的氨基酸序列与葡萄ARF8的相似性最高(83%),与番茄ARF8的亲缘关系最近.利用实时荧光定量PCR技术,检测该基因在茶树越冬芽休眠到萌发后不同时期的表达情况.结果显示 CsARF1在茶树越冬芽深休眠和萌动期表达量较高,表明该基因与茶树越冬芽的休眠维持及解除密切相关.
Cloning and Expression Analysis of Auxin Response Factor Gene (CsARF1) in Tea Plant (Camellia sinensis [L.] O. Kuntze)
Auxin response factors (ARFs) are transcription factors that bind to TGTCTC auxin response elements in promoters of early/primary response genes and regulate the expression of auxin response genes. The full-length cDNA of one ARF gene named CsARF1 (GenBank accession number JX307853) was firstly cloned from tea plant (Camellia sinensis [L.] O. Kuntze) by SMART-RACE-PCR. The results indicated that the full-length cDNA of CsARF1 was 3222 bp containing 2463 bp ORF which encoded 820 amino acid residues with a putative molecular mass of 49.35 kD. The soluble protein encoded by CsARF1 functioned in the cytoplasm and consisted of an amino-terminal DNA-binding domain (B3), a carboxy-terminal dimerization domain (IAA_ARF), and a Gln, Ser and Leu-rich middle region, which is proposed to function as an activation domain. Blast and phy-logenetic analysis showed that the protein encoded by CsARF1 shared the highest identity (83%) with ARF8 in Vitis vinifera, and had close genetic relationship with ARF8 in Solanum lycopersicum. The expression analysis of CsARF1 conducted by qRT-PCT during the different phases of bud dormancy and bud break indicated that CsARF1 had a marked rise in the expression level at deep dormant stage and sprouting stage, demonstrating that CsARF1 is relevant to the regulation of tea plant bud dormancy and bud break.
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