霍山石斛DhGMDS基因克隆及低温胁迫下表达  

Cloning of DhGMDS Gene from Dendrobium huoshanense and Its Expression under Low Temperature Stress

作　　者：吴丽萍 苏兴隆 王兆健 俞年军 彭代银 邢世海 WU Liping;SU Xinglong;WANG Zhaojian;YU Nianjun;PENG Daiyin;XING Shihai(College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China;2Traditional Chinese Medicine Resources Protection and Development, Anhui Academy of Chinese Medicine, Hefei 230012, China)

机构地区：[1]安徽中医药大学药学院,合肥230012 [2]安徽省中医药科学院中药资源保护与开发研究所,合肥230012

出　　处：《西北植物学报》2022年第2期221-228,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金区域联合基金(U19A2009);安徽省留学人员创新项目择优资助计划(DT1810003);安徽省教育厅高等学校自然科学研究项目(KJ2019A0453);安徽省科技厅自然科学基金(1908085MH268);安徽中医药大学教学改革重点项目(2019xjjy_zd020)。

摘　　要：基于霍山石斛转录组数据库,利用染色体步移技术,克隆霍山石斛GDP-甘露糖4,6-脱水酶基因(DhGMDS)及其启动子,采用实时荧光定量PCR方法检测该基因的组织表达模式及低温处理下的表达,为进一步解析霍山石斛抗寒机制和遗传改良提供理论依据。结果表明:(1)成功克隆获得DhGMDS基因(GenBank登录号MW855573),其cDNA序列为1134 bp,gDNA序列为1523 bp,无内含子。(2)序列一致性分析显示,DhGMDS蛋白与黄花石斛(Dendrobium catenatum)的GMDS蛋白序列相似性达到99.03%;系统进化树分析表明,霍山石斛DhGMDS基因与黄花石斛GMDS基因处于同一进化节点上,二者亲缘关系近。(3)启动子序列分析发现,在DhGMDS基因上游启动子区含有光、低温、干旱和ABA响应元件,且具有MYB等转录因子识别和结合位点。(4)实时荧光定量PCR分析显示,DhGMDS基因在霍山石斛的花中表达量最高,其次为茎、叶,根中表达量最低;经4℃低温处理72 h后,DhGMDS基因受低温胁迫诱导上调表达,且在低温处理24 h的相对表达量最高,表明DhGMDS基因参与了植物逆境响应过程。研究推测,低温对DhGMDS基因的诱导表达可能依赖于上游MYB转录因子的调控。Based on the transcriptome database of Dendrobium huoshanense,we cloned GDP mannose 4,6-dehydratase gene(DhGMDS)and the promoter sequence by Genome Walking technique.The tissue expression pattern and expression under low temperature treatment were detected by real-time quantitative PCR,so as to provide a theoretical basis for further analyzing the cold resistance mechanism and genetic improvement of D.huoshanense.Results showed that:(1)DhGMDS gene(GenBank accession number MW855573)was successfully cloned,and its cDNA sequence of the DhGMDS gene was 1134 bp,and the gDNA sequence was 1523 bp,without introns.(2)Sequence similarity showed that the identity of DhGMDS protein and GMDS protein of D.catenatum was 99.03%;Phylogenetic tree analysis showed that DhGMDS gene of D.huoshanense and GMDS gene of D.catenatum were in the same evolutionary node,and they had the relatively close genetic relationship.(3)The promoter sequence analysis showed that the upstream promoter region of DhGMDS gene contained light response,low temperature,drought and ABA response elements,MYB transcription factor recognition and binding sites,etc.(4)qRT-PCR analysis showed that the expression level of DhGMDS gene was the highest in flowers,followed by stems and leaves,and the lowest in roots.After 72 h of low temperature treatment at 4℃,DhGMDS gene was up-regulated by low temperature stress,and the relative expression was the highest at 24 h,indicating that the DhGMDS gene was involved in the process of plant stress response.Studies have speculated that the induction of DhGMDS gene expression by low temperature may depend on the regulation of the upstream MYB transcription factor.

关 键 词：霍山石斛 GDP-甘露糖4 6-脱水酶 启动子 低温胁迫 基因表达 

分 类 号：Q949.718.43[生物学—植物学] Q785