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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Frigi-da-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 DlUP-5 cDNA sequence of the longan embryo-related unknown protein gene was cloned by degenerate primer combined with rapid cDNA cloning (RACE) using the embryogenic culture of longan somatic embryo. The open reading frame (ORF) consisting of 681 nucleotides and encoding a polypeptide of 226 amino acids (GenBank accession number: GQ443759) was 887 bp in length, with a molecular weight of 24511.9 u and a theoretical pI of 9.65. Is a member of the Frigi-da-like family of proteins and is a hydrophilic protein with a Frigida assembly, a typical signal peptide structure, and no transmembrane helices. Irregular curling is the largest structural element of the family of proteins and is mainly concentrated in the C-terminal region The results of real-time quantitative PCR showed that the gene was expressed in the longan somatic embryogenesis with the tendency of “N ” shape, with the incomplete embryonic compact structure stage being the highest, while the torpedo-shaped embryo stage expression The results of ANOVA showed that the expression of DlUP-5 gene in torpedo-shaped embryos had extremely significant difference with other seven stages.The results of this study have a preliminary understanding of the role of DlUP-5 in somatic embryogenesis of longan, Its in the dragon Embryonic callus aspects of embryogenesis ability, early normal development of somatic embryos and somatic embryo maturation process, etc. may play an important role.