山药SuSy基因全长cDNA序列的结构、进化和表达

Structure,Evolution and Expression of a Full-Length cDNA Sequence of SuSy Gene from Yam

【目的】阐明山药蔗糖合成酶(SuSy,EC 2.4.1.13)基因家族的成员及其功能。【方法】利用RT-PCR和RACE技术从大薯(Dioscorea alata)地下块茎中分离到1个SuSy基因(DaSuSy1)全长cDNA序列,并用DANSTAR和Clastal W等软件分析该序列的结构特征和分子进化,采用RT-PCR和Northern杂交对该基因的时空表达进行分析。【结果】DaSuSy1全长cDNA序列大小为2673bp,其中最大开放阅读框(ORF)、5'端和3'端的非翻译区分别含有2445bp、7bp和221bp,而且3'端的非翻译区含1个24nt的Poly(A+)尾;最大ORF可编码814个氨基酸,分子量为92.76kD,等电点为6.42,含有蔗糖合成酶和葡糖基转移酶两个保守功能域及两个磷酸化位点,即N端的Ser10和C端的SNLDRRET781 RR(Ser774～Thr781)。该基因在全长cDNA序列、编码区cDNA序列及其编码氨基酸序列的水平上与GenBank中所选已知物种SuSy基因相应序列的同源性分别达45.3%～71.3%、45.8%～74.8%和50%～84.7%,与禾本科植物SuSy基因家族中一些成员亲缘关系最近。DaSuSy1在非光合器官中表达,其中地下块茎表达最为强烈,而且从块茎形成初前期开始表达一直增强至盛中期,此后逐渐减弱。【结论】DaSuSy1是山药SuSy基因家族成员,归为单子叶植物SuSy基因的组别,仅在非光合器官中表达编码负责蔗糖-淀粉转化功能的SuSy同工型。

[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 （DaSuSyl） 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] DaSuSyl 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 Ser^10 at N-end of and SNLDRRETTM RR（Ser^774-Thr^781） at C-end of amino acid sequence. At the levels of complete eDNA sequences, coding regions and its deduced amino acid sequences, DaSuSyl 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. DaSuSyl 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 undergrotmd tuber bulking, and thereafter gradually decreased. [Conclusion] DaSuSyl 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）.