以龙眼体胚发生早期的胚性培养物为材料,通过简并引物结合PCR进行cDNA末端快速克隆(RACE)的技术,克隆到了龙眼体胚相关未知蛋白基因DlUP-5全长序列,其cDNA全长为887 bp,由681个核苷酸组成的开放阅读框,编码226个氨基酸(GenBank登陆号为GQ443759).该基因推导的蛋白分子质量为24511.9 u,理论等电点pI为9.65；该蛋白是Frigida-like蛋白质家族成员,是一个具有Frigida组件,无典型信号肽结构,无跨膜螺旋的亲水性蛋白；不规则卷曲是其最大量的结构元件,并且主要集中在C端区域.实时荧光定量PCR分析结果显示,该基因在龙眼体胚发生过程中均有表达,呈“N”形趋势,其中以不完全胚性紧实结构阶段最高,而鱼雷形胚阶段表达量最低；方差分析表明,鱼雷形胚阶段DlUP-5基因表达量与其他7个阶段存在极显著差异.该研究结果对DlUP-5基因在龙眼体胚发生过程中的作用有了一个初步的认识,其在龙眼胚性愈伤组织胚胎发生能力、早期体胚正常发育和体胚成熟过程等方面可能起重要作用.%The full-length sequence of the gene DlUP-5 (Dimocarpus longan unknown protein gene 5) from embryogenic cultures was obtained by using degenerate oligonucleotide primers together with rapid amplification of cDNA ends (RACE) technique. The full-length cDNA sequence of DlUP-5 gene is 887 bp. It contained a 681-nucleotides-long open reading frame (ORF) which encoded a protein of 226 amino acid residues (GenBank No. GQ443759). Bioinformatics analysis showed that the molecular weight of DlUP-5 protein was 24511.9 u with pi value 9.65. The protein, including a Frigida module, belongs to Frigida-like family. It has hydrophilic characteristics and no transmembrane properties as well as no signal peptide. Random coils are the most maximum structural elements in it and were found mainly concentrating in the C terminal domain. The expression of DlUP-5 gene was also observed during longan somatic embryogenesis. Its expression trend was "N" shape, the highest expression level occurred at incomplete compact pro-embryogenic cultures stage, and the lowest appeared at torpedo embryos stage. The variance analysis results showed that the relative mRNA transcription levels of DlUP-5 at torpedo embryos stage was significantly higher than those at the other seven stages. It shaded light on the result suggest that the DlUP-5 gene has some important functions during longan somatic embryogenesis. According to the transcription level change of DlUP-5, it may play some important functions in maintaining high embryogenesis competence of longan embryogenic callus and facilitate further development and growth of embryos. This result laid a foundation for the further study on the function of DlUP-5 gene during longan somatic embryogenesis.
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