龙眼体胚发生相关未知蛋白DlUP-3基因的克隆与表达分析

Cloning and Expression Analysis of Unknown Protein DlUP-3 Gene Related to Somatic Embryogenesis in Dimocarpus longan Lour.

在龙眼体胚发生早期的蛋白质组学研究中,发现1个体胚发生相关未知蛋白DlUP-3,通过简并引物结合RACE技术进行其基因全长序列克隆。结果显示:(1)克隆到的龙眼体胚发生相关未知蛋白基因DlUP-3的全长cDNA序列为1 681bp,开放阅读框由1 017个核苷酸组成,编码338个氨基酸(GenBank登录号为GQ167202)。(2)生物信息学分析发现,该基因推导蛋白分子量为36 854.2Da,pI为9.05;该蛋白为Ras蛋白质家族成员,具有ATP/GTP-binding site motif A(P-loop)结合位点和1个典型的Ras_like_GTPase superfamily组件,无典型信号肽结构,但有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,散布于整个蛋白质中。(3)实时荧光定量PCR分析显示,该基因在龙眼体胚发生过程中均有表达,其中以胚性愈伤组织阶段表达量最低,而球形胚阶段最高。研究表明,DlUP-3基因在龙眼体胚发生过程尤其是球形胚阶段有重要的作用,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。

An unknown protein gene related to somatic embryogenesis had been discovered and named Di- mocarpus longan unknown protein gene 3 （DlUP-3） during the proteomic analysis of early somatic embry- ogenesis in longan. It was cloned by using degenerate oligonucleotide primers together with RACE. The full-length cDNA sequence of DlUP-3 gene was 1 681 bp. It contains a 1 017-nucleotides-long open reading frame （ORF） which encoded a protein of 338 amino acid residues （GenBank No. GQ167202）. Bioinformat- ics analysis showed that the molecular weight of D1UP-3 protein is 36 854.2 Da with pI 9.05 ,it is a mem- ber of the Ras protein superfamily,which has ATP/GTP-binding site motif A （P-loop） binding site,Ras_ like GTPase superfamily module, hydrophilic characteristics and transmembrane properties but no signal peptide. Random coils are the most maximum structural elements and were found spreading in the whole polypeptide chain. The expression of DlUP-3 gene was also investigated during longan somatic embryogen- esis according to the results of real-time quantitative PCR. Its expression was the lowest at the the friable- embryogenic callus stage and the highest at globular embryos stage. There had an elementary understand- ing to the important function of DlUP-3 gene during longan somatic embryogenesis, especially at globular embryos stage. This result laid a foundation for the further study on the function of DlUP-3 gene during longan somatic embryogenesis.