关键词: 大薯(Dioscorea alata)')">大薯(Dioscorea alata), 蔗糖合成酶, 基因克隆, 结构特征, 分子进化, 表达分析

Abstract:

【Objective】 The purpose of this paper is to elucidate sucrose synthase (SuSy, EC 2.4.1.13) gene family members and their functions. 【Method】 Both RT-PCR and RACE technique were employed to isolate a full-length cDNA sequence of SuSy gene (DaSuSy1) from the underground tubers of yam (Dioscorea alata), both structural characteristics and molecular evolution of which were analyzed with such software as DANSTAR and Clastal W, and the spatial and temporal expressions of this gene were characterized by RT-PCR and Northern hybridization. 【Result】 DaSuSy1 full-length cDNA sequence was 2 673 bp in size, which comprised a 2 445 bp largest open reading frame (ORF), a 7 bp 5′ noncoding region and a 221 bp 3′ noncoding region with a 24 nt poly (A+) tailor. A polypeptide of 814 amino acids with a 92.76 kD molecular weight and a theoretical pI of 6.42 was putatively encoded by the largest ORF, containing both two conserved functional regions as sucrose synthase and glucosyl-transferase and two phosphorylated sites as Ser10 at N-end of and SNLDRRET781 RR(Ser774-Thr781) at C-end of amino acid sequence. At the levels of complete cDNA sequences, coding regions and its deduced amino acid sequences, DaSuSy1 shared the similarities of 45.3%-71.3%, 45.8%-74.8%, 50%-84.7%, respectively, with SuSy genes of the selected and known species in GenBank, and had the closest genetic relationship with some members of SuSy gene families of gramineous plants. DaSuSy1 expressed only in non-photosynthetic organs of yam was not only strongly expressed in the underground tubers, but also its expression abundances progressively increased from the initiation of early stage to the part of middle stage of the underground tuber bulking, and thereafter gradually decreased. 【Conclusion】 DaSuSy1 belonging in yam SuSy gene family members was assigned to the monocot SuSy gene group, which was expressed to encode SuSy isoform responsible for the conversion of sucrose to starch only in non-photosynthetic organs of yam (D. alata).

Key words: yam (Dioscorea alata)')">yam (Dioscorea alata), sucrose synthase, gene cloning, structural characteristics, molecular evolution, expression analysis