山药赤霉素受体基因DoGID1A的克隆及表达分析  

Cloning and Expression Analysis of Gibberellin Receptor Gene DoGID1A in Dioscorea opposita

作　　者：龙雯虹[1] 孟金贵[1] 徐升胜[1] 张雪梅[2] 段延碧[3] 杨荣萍[1] LONG Wen-Hong;MENG Jin-Gui;XU Sheng-Sheng;ZHANG Xue-Mei;DUAN Yan-Bi;YANG Rong-Ping(College of Horticulture and Landscape,Yunnan Agricultural University,Kunming 650201,China;College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;Experiment Teaching Center for Agriculture Specialty,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学园林园艺学院,昆明650201 [2]云南农业大学农学与生物技术学院,昆明650201 [3]云南农业大学农科教学实验中心,昆明650201

出　　处：《农业生物技术学报》2019年第11期1933-1941,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31460322)

摘　　要：赤霉素受体(gibberellin insentive dwarf 1,GID1)是赤霉素(gibberellin,GA)信号转导途径的重要元件,直接影响着赤霉素对植物的作用效果。以山药(Dioscorea opposita)品种'牛尾山药'珠芽的表皮为料材,采用反转录PCR(reverse transcription-polymerase chain reaction,RT-PCR)和cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆山药的赤霉素受体基因GID1的cDNA全长,用qRTPCR检测山药GID1基因在山药珠芽采后不同时期的表达量,及其对多效唑处理的响应。结果显示,克隆得到的山药赤霉素受体基因GID1的cDNA全长1123 bp,开放阅读框996 bp,编码331个氨基酸,命名为DoGID1A(GenBank No.MK268684)。生物信息学分析显示,DoGID1A蛋白质分子量为3.72×104 D,理论等电点为8.25,为不稳定蛋白,此蛋白无跨膜结构和信号肽,有典型的自水解酶(abhydrolase)活性区域(93~304 aa)。蛋白质同源性分析表明,DoGID1A蛋白与小果野芭蕉(Musa acuminata)等13种植物的GID1蛋白整体同源性达73.56%,有多个高度保守的区域,具有与DELLA和GA结合的活性区域。系统进化分析显示,山药和水稻(Oryza sativa)的GID1蛋白与其他植物进化关系远,这两种植物的GID1蛋白各自为一类,其他植物则是单子叶植物聚于一类,双子叶植物聚于另一类。qRT-PCR分析显示,山药珠芽发芽期DoGID1A表达量升高,多效唑处理使该基因表达量上调。预测该基因与山药珠芽休眠解除及发芽有关,本研究结果为进一步研究该基因的表达规律和功能提供了基础资料。Gibberellin receptor GID1(gibberellin insentive dwarf 1),one of important elements in gibberellin(GA)signal transduction pathway,directly affects the effect of gibberellin on plant.A cDNA sequence encoding gibberellin receptor GID1 was cloned from epidermis of bulbils of'Niuwei Yam'(Dioscorea opposita)with reverse transcription-polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE)techniques.The expression of the cloned yam GID1 gene at different stages after bulbils were harvested and the response of the gene expression to paclobutrazole were tested by qRT-PCR.The results showed that the obtained gene had a full length of 1123 bp with 996 bp opening reading fram encoding 331 amino acids,and named DoGID1A(GenBank No.MK268684).Bioinformation analysis showed that the molecular weight and theoretic isoelectric piont of the protein was 3.72×104 D and 8.25,respectively.The DoGID1A protein was unstable protein with an abhydrolase domain(93~304 aa)but without transmembrance domain or signal peptide.Homologous alignment showed that DoGID1A protein shared 73.56%sequence similarity with 13 specieces including Musa acuminata,and shared conserved many domains including motifs linking hormone GA and protein DELLA.Phylogenetic tree showed all dicotyledons were cluster in a group,monocotyledonous plants except rice(Oryza sativa)and yam shared another group.Yam was clustered out alone,so was rice.qRT-PCR analysis showed the expression of DoGID1A gene increased during yam bulbil sprouting and was enhanced by paclobutrazol treatment.These results indicated that DoGID1A gene could be associated with yam bulbil sprouting.This study provides basic information for further research on expression pattern and function of this gene.

关 键 词：山药 赤霉素受体GID1 基因克隆 基因表达 

分 类 号：S632.1[农业科学—蔬菜学]