龙眼胚性愈伤组织DRM1基因的克隆及其定位与表达分析  

Cloning,Localization and Expression Analysis of DRM1 Gene from Embryogenic Calli of Dimocarpus longan Lour.

作　　者：白玉[1] 陈晓慧[1] 谢礼洋[1] 吴晓佩 林玉玲[1] 赖钟雄[1] BAI Yu;CHEN Xiaohui;XIE Liyang;WU Xiaopei;LIN Yuling;LAI Zhongxiong(Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, China)

机构地区：[1]福建农林大学园艺植物生物工程研究所,福州350002

出　　处：《西北植物学报》2017年第11期2097-2105,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31572088;31672127);福建省重大专项(2015NZ0002-1);福建省自然科学基金杰出青年项目(2015J06004);福建省新世纪人才项目(20151104);福建农林大学"杰出青年科研人才"培养专项基金(xjq201405)

摘　　要：该研究以龙眼胚性愈伤组织的转录组数据为基础,对龙眼胚性愈伤组织DlDRM1基因进行克隆和生物学信息分析,并检测其在体胚发生过程中不同发育阶段、不同浓度的外源激素(2,4-D、IAA、KT)处理下及不同组织部位的表达,以揭示DlDRM1基因在龙眼体胚发生过程中的功能。结果表明:(1)从龙眼转录组unigene序列筛选获得龙眼结构域重排甲基化酶1基因(命名为DlDRM1)全长序列,并利用RT-PCR法从‘红核子’龙眼胚性愈伤组织中克隆获得DlDRM1基因的cDNA全长序列(GenBank登录号为KY990493);DlDRM1基因cDNA全长2 574bp,包括494bp的5′UTR,184bp的3′UTR,可编码包含631个氨基酸的蛋白质。(2)生物信息学分析显示,DlDRM1是一个不稳定的亲水蛋白,不含信号肽,不存在跨膜结构域,其分子式为C_(3104)H_(4839)N_(851)O_(984)S_(28);序列比对和系统进化分析表明,龙眼DlDRM1与脐橙DRM相似度最高(76.85%),二者亲缘关系也最为接近。(3)实时荧光定量PCR分析发现,DlDRM1在龙眼各组织器官中均有表达,且在果肉中表达量最高,其次是花蕾,在叶中的表达量最低;DlDRM1基因在非胚性愈伤组织中表达量最高,而且在非胚性愈伤向胚性愈伤转变过程中DlDRM1基因的表达量呈逐步下降趋势,说明DlDRM1基因与体胚胚性呈负相关关系,在龙眼体胚发生过程中可能发挥着重要的作用;一定浓度的IAA和2,4-D能够促进DlDRM1基因的表达,而KT则抑制DlDRM1的表达。(4)亚细胞定位结果表明,DlDRM1定位于细胞核和细胞膜上。Based on the transcriptome data of longan embryogenic callus,we cloned the Dimocarpus longan DRM1gene.The functions of DlDRM1was analyzed by means of bioinformatics and real-time quantitative PCR.Expression of DlDRM1was analyzed in the stages of somatic embryogenesis,exogenous hormone(2,4-D,IAA,KT)treatment and different tissues in D.longan.The purpose is to reveal the D.longan DRM1gene function in the process of longan somatic embryos.The results showed that:(1)the full-length sequence of longan s Domains Rearranged Methyltransferase1gene(DlDRM1)was successfully cloned from embryogenic callus of‘Honghezi’longan by using RT-PCR(GenBank accession number is KY990493)based on the transcriptome data of longan embryogenic callus unigene sequences.The cDNA full-length is2574bp,including494bp5′UTR and184bp3′UTR,and it could encodes protein with631amino acids.(2)Bioinformatics analysis showed that DlDRM1(molecular formula:C3104H4839N851O984S28)was an unstable hydrophile protein,without signal peptide and transmembrane domain.Sequence alignment result showed DlDRM1was highly similar to DRM in navel orange(up to76.85%),and the closest genetic relationship was also found between the two proteins according to phylogenetic analysis.(3)Quantitative real-time PCR showed that DlDRM1expresses in all of the tissues and organs in longan and the highest expression was found in flesh,followed by flower buds,and the lowest expression was found in leaves.Notably,the expression of DlDRM1in non-embryonic callus was found to be significantly higher than that in embryonic callus,and its expression decreases during the process of the non embryonic callus changes to be embryonic.These indicated that DlDRM1was negatively correlated with the embryonic of somatic embryo and it might contribute to the longan somatic embryogenesis.Moreover,we also studied the effects of hormones on the expression of DIDRM1.And it was found that certain concentrations of IAA and2,4-D could promote its expression,while KT inhibit its expression.(4)Subcellul

关 键 词：龙眼 体胚发生 DlDRM1 基因克隆 表达分析 

分 类 号：Q785[生物学—分子生物学] Q786