文心兰乙烯不敏感基因EIN2的克隆及表达分析  

Cloning and Expression of Ethylene Insensitivity Gene EIN2 in Oncidesa

作　　者：时欢 李蓉[1] 高玉莹 林玉玲[1] 杜宜殷 赖钟雄[1] 黄鹏林 SHI Huan;LI Rong;GAO Yuying;LIN Yuling;DU Yiyin;LAI Zhongxiong;HUANG Pengling(Institute of IIorticulural Biotechnology,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Department of Ilorticulture ~ Landscape Architecture,Taiwan University,Taipei 10617,China;Graduate Institute of Biotechnology,Chinese Culture University,Taipei 11114,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 [2]台湾大学园艺暨景观学系,台北10617 [3]中国文化大学生物科技研究所,台北11114

出　　处：《西北植物学报》2018年第9期1613-1619,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：福建省重大专项(2015NZ-0002-1)

摘　　要：该研究采用PCR、RACE方法,对文心兰‘南茜'的乙烯不敏感蛋白基因(ethylene insensitive 2,EIN2)进行克隆及生物信息学分析,并采用qRT-PCR技术,对该基因在文心兰不同组织器官和不同花期中的表达模式进行分析。结果表明:(1)成功克隆得到文心兰EIN2基因序列,命名为OnEIN2(MH497388);该基因cDNA序列全长为4 177bp,其中开放阅读框3 879bp,编码1 292个氨基酸,3′非编码区长208bp,5′非编码区长90bp。(2)生物信息学分析显示OnEIN2是一个不稳定的疏水蛋白,含有跨膜结构,分子式为C6406H9988N1670O1897S47;蛋白质分子量142.22kD,理论等电点5.80。多序列比对和系统进化分析表明,文心兰EIN2与铁皮石斛EIN2的相似度最高(81.98%),二者亲缘关系也最为接近。(3)实时荧光定量PCR分析发现,OnEIN2基因在根、茎、叶花中均有表达,在花中表达量最高,茎中表达量最低;而在不同花期中,盛开期表达量最高,其次是衰老期。研究表明,文心兰OnEIN2基因在开花和衰老过程中可能有重要的作用。In this experiment,PCR and RACE methods were used to clone and bioinformatics was used to analyze the ethylene insensitive 2（EIN2）gene of Oncidesa Gower Ramsey.The qRT-PCR technique was used to analyze the different tissue sites and different flowering stages of Oncidesa.The results showed that：（1）The EIN2 gene of Oncidesa,named OnEIN2（MH497388）was successfully cloned;the cDNA of OnEIN2 full-length was 4 177 bp,contains a 3 879 bp open reading frame（ORF）,encoding 1 292 amino acids,the non-coding region of 3′and 5′were 208 bp and 90 bp.（2）Bioinformatics analysis showed that OnEIN2（molecular formula：C6406 H9988 N1670 O1897 S47）was an unstable hydrophobin protein containingatransmembrane domain;the molecular weight of the protein was 142.22 kD and the theoretical isoelectric point was 5.80.Multiple sequence alignment and phylogenetic analysis showed that the similarity of OnEIN2 was highly similar to EIN2 in Dendrobium catenatum（up to 81.98%）,and the genetic relationship between them was the closest.（3）Quantitative real-time PCR showed that OnEIN2 expresses in roots,stems,leaves and flowers;with the highest expression level in flowers and the lowest level in stems,while the expression level was the highest in different flowering stages during the blooming period,followed by senescence stage.This study showed that OnEIN2 may play an important role in flowering and senescence.

关 键 词：文心兰 EIN2 克隆 表达分析 

分 类 号：Q785[生物学—分子生物学] Q786