苹果SUPERMAN类蛋白家族的基因克隆与生物信息学分析

摘要

SUPERMAN family genes are important transcription factors in the plant growth and development. In order to understand physiological functions of SUPERMAN family genes in apple (Malus domestica Borkh.), the MdLIFa (GenBank accession No. HM3 70493) and its promoter were cloned using RT-PCR and anchored PCR from apple cultivar Fuji (M. Domestica. Cv. Fuji). The 11 genes of SUPERMAN family in apple were cloned by PCR amplification using gene-specific primers. All 12 genes were designated as MdLIFa/MdLIFb (GenBank accession No. HQ260429), MdSUPla (HQ4W477)/MdSUPlb (HQ418478), MdSUP4(KQ418475) /MdSUPH (HQ418476), MdSUP5a (E.Q418469)/MdSUP13b (HQ418470), MdSUP9a(HQ4l8473)/MdSUP9b (HQ418474), and MdSUP5b (HQ418471)/MdSUP13a (HQ418472), located on chromosomes 11/3, 1/1, 4/12,5/13, 9/unknown, and 5/13, respectively. The similarity of deduce amino acid sequences of each pair of genes was higher than 86%. The subcellular localization of MdLlFa was transient expressed in the cell nucleus by the particle bombardment into onion (Allium cepa) epidermal cells. Cis-acting elements, such as POLLEN1LELAT52 (AGAAA), GTGANTG10 (GTGA), ROOTMOTIFTAPOX1 (ATATT), DOFCOREZM (AAAG), and GT1 CONSENSUS (GRWAAW), were found in the upstream of MdLlFa. Phylogenetic analysis showed that MdLIFa/b were clustered in one group, together with MdSUP9a/9b and MdSUP5a/13b while MdSUP1aJb, MdSUP5b/MdSUP13, and MdSUP4/12 in another group. These genes were putatively involved in plant morphogenesis and floral organogenesis. The SUPERMA N family genes in apple are highly homologous in pair with diverse biological functions, which provide useful information for further gene characterization and genetic transformation.%SUPERMAN类基因是植物生长发育中的重要转录因子.为了揭示苹果SUPERMAN类蛋白家族的生理基因功能,本研究以富士苹果(Malus domestica Borkh.cv.Fuji)为试材,利用RT-PCR方法和锚定PCR方法克隆了MdLIFa(GenBank登录号HM370493)和启动子序列,利用特异引物PCR方法克隆了苹果基因组中其余11个SUPERMAN类基因.12个基因命名为MdLIFa/MdLIFb(GenBank登录号:HQ260429)、MdSUP1a(HQ418477)/MdSUP1b(HQ418478)、MdSUP4(HQ418475)/MdSUP12(HQ418476)、MdSUP5a(HQ418469)/MdSUP13b(HQ418470)、MdSUP9a(HQ418473)/MdSUP9b(HQ418474)和MdSUP5b(HQ418471)/MdSUP13a(HQ418472),分别位于11/3、1/1、4/12、5/13、9/未知和5/13号染色体,每对基因间的同源性均在86％以上.通过基因枪法轰击洋葱(Allium cepa)表皮细胞,基因瞬时表达证明MdLIFa定位于细胞核内.MdLIFa基因启动子序列中含有多个参与花粉发育、根特异性表达、与光诱导和Dof单锌指基因家族作用的调控元件等.系统进化分析表明,MdLIFa/b、MdSUP9a/9b和MdSUP5a/13b、以及MdSUP1a/b、MdSUP5b/MdSUP13a和MdSUP4/12分别聚类为一组,参与植株的形态建成和花器官发育.苹果12个SUPERMAN类基因成对高度同源,具有生物学功能的多样性,为深入的基因鉴定和遗传转化提供有用信息.