龙眼体胚相关未知蛋白基因DlUP-4的克隆与表达分析

Cloning of the Gene Encoding a Unknown Protein DlUP-4 Related to Somatic Embryogenesis from Embryogenic Cultures and Its Quantitative Expression during Somatic Embryogenesis in Longan(Dimocarpus longan Lour.)

以龙眼(Dimocarpus longan Lour.)胚性培养物为材料,采用简并引物结合RACE方法分离得到龙眼体胚相关未知蛋白基因DlUP-4的cDNA全长序列,全长为1 061 bp,由735个核苷酸组成的开放阅读框,编码244个氨基酸(GenBank登录号为GQ443758)。采用生物信息学方法分析,发现该基因推导的蛋白分子量为28 576 ku,pI为9.96;该蛋白为60S Ribosomal protein L30蛋白质家族成员中的L7蛋白,是一个具有核糖体蛋白L30信号位点和1个典型的Ribosomal L30 like superfamily组件,无典型信号肽结构,无跨膜螺旋的亲水性蛋白;α螺旋是该蛋白最大量的结构元件,散布于整个蛋白质中。实时荧光定量PCR分析结果显示,DlUP-4基因在龙眼体胚发生过程中均有表达,其表达水平的趋势呈"M"形,其中2个高峰出现在不完全胚性紧实结构阶段和鱼雷形胚阶段,而球形胚阶段最低。该研究结果对DlUP-4基因在龙眼体胚发生过程中的作用有了一个初步的认识,其在龙眼体胚发生过程中,在调控体胚发生的启动、细胞的极化、细胞的增殖等方面可能有重要的功能,这为进一步研究该基因在龙眼体胚发生过程中的功能奠定了基础。

The full-length sequence of a Dimocarpus longan unknown protein gene 4（DlUP-4）was cloned from embryogenic cultures during the early longan somatic embryogenesis by using degenerate oligonucleotide primers together with RACE technique.The full-length cDNA sequence of DlUP-4 gene was 1 061 bp（GenBank No.GQ443758）.It contained a 735-nucleotides-long open reading frame（ORF）which encoded a protein of 244 amino acid residues.Bioinformatics analysis showed that the molecular weight of DlUP-4 protein was 28.576 ku with pI value 9.96,it is a L7 protein of the 60S ribosomal protein L30 superfamily,which had a ribosomal protein L30 single site,ribosomal L30 like superfamily module,hydrophilic characteristics and no transmembrane properties as well as no signal peptide.Alpha helix were the most maximum structural elements and were found spreading in the whole polypeptide chain.The expression of DlUP-4 gene was also observed during longan somatic embryogenesis.Its expression trend was ＂M＂ shape,the two highest expression levels occurred at incomplete compact pro-embryogenic cultures stage and the topedo embryos stage,and the lowest appeared at the globular embryos stage.There had a preliminary knowledge about the important function of DlUP-4 gene during longan somatic embryogenesis.It may serve some important functions,and those were the regulation of somatic embryo initiation,cell polarization and cell proliferation,etc.This results laid a foundation for the further study on the function of DlUP-4 gene during longan somatic embryogenesis.