中文摘要：

以龙眼松散型胚性愈伤组织为材料,采用RT-PCR和RACE技术克隆获得生长素响应因子ARF5a cDNA全长序列,利用实时荧光定量法检测其在龙眼体胚不同发育时期的转录水平,并将其与绿色荧光蛋白（GFP）构建成亚细胞定位载体侵染洋葱表皮细胞。结果表明：龙眼ARF5a（命名为DlARF5a,GenBank登录号为KF739401）的cDNA序列全长为3 322 bp,其中开放阅读框为2 829 bp,212 bp 5′非编码区,258 bp 3′非编码区,编码942个氨基酸。生物信息学预测显示,DlARF5a编码蛋白具有B3、Auxin-resp和AUX-IAA保守区与结构功能域,中间区域富含谷甘氨酸、丝氨酸和亮氨酸,是一个定位于细胞质的具有促进转录活性功能的水溶性蛋白。系统进化树分析表明,该基因编码的氨基酸序列与大豆的亲缘关系最近。qRT-PCR检测显示,DlARF5a在龙眼球形胚和鱼雷形胚时期表达显著增强,而在6个发育阶段中子叶形胚的表达量最低,推测DlARF5a参与龙眼体胚中鱼雷胚时期的形态建成。共聚焦显微镜观察结果显示,未添加外源生长素IAA的DlARF5a蛋白定位于细胞质中,而经外源生长素处理的DlARF5a蛋白在细胞膜、细胞质和细胞核中均有表达,推测龙眼生长素响应蛋白DlARF5a能够响应外源生长素IAA而改变其在细胞中的空间位置。

英文摘要：

The full-length cDNA of one ARF gene named DlARF5a（GenBank accession number：KF739401）was cloned from embryogenic callus of Dimocarpus longan by the RT-PCR combined with RACE method.Then the mRNA transcription level of the gene in the process of somatic embryogenesis was determined by qRT-PCR（real-time reverse transcription PCR） method.And the expression of ARF5a-GFP fusion protein in onion epidermal cells revealed its subcellular localization.The results indicated the complete cDNA sequence of DlARF5a was 3 322 bp containing 2 829 bp ORF encoding 942 amino acid,with 5′ and 3′ untranslated regions（UTR） of 212 bp and 258 bp,respectively.Bioinformatics analysis suggested that DlARF5a was probably a soluble protein,and functioned in the cytoplasm and consisted of a B3,Auxin-resp and AUX-IAA conservative district and structural function domain,and a Gln,Ser and Leu-rich middle region,which was considered as an activation domain.Phylogenetic analysis showed that protein encoded by DlARF5a had close genetic relationship with ARF5 in Citrus sinensis.The qPCR detection showed that DlARF5a had a remarkable rise in the expression levels at globular embryo and torpedo embryo stages,but the nadir mRNA transcription level of DlARF5a gene occurred at the cotyledonary stage（stage 6）,demonstrating that DlARF5a was relevant to the torpedo embryo morphogenesis during longan somatic embryogenesis.The subcellular localization assays showed that the DlARF5a protein was located in the cytoplasm without adding the exogenous auxin IAA by confocal microscopy.Whereas,DlARF5a protein was located in the cell membrane,cytoplasm and nucleus under 1.0 mg/L IAA treatment,wich can be speculated that DlARF5a protein was able to change its spatial location in the cells in response to the external IAA.