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园艺学报 ›› 2018, Vol. 45 ›› Issue (8): 1501-1512.doi: 10.16420/j.issn.0513-353x.2018-0138

• 研究论文 • 上一篇    下一篇

龙眼体胚发生早期miR166初级体的克隆与表达分析

张清林,苏立遥,厉 雪,张舒婷,徐小萍,陈晓慧,王培育,李 蓉,张梓浩,陈裕坤,赖钟雄*,林玉玲*   

  1. 福建农林大学园艺植物生物工程研究所,福州 350002
  • 出版日期:2018-08-25 发布日期:2018-08-25
  • 基金资助:
    福建省自然科学基金杰出青年项目(2015J06004);国家自然科学基金项目(31672127,31572088);福建农林大学科技创新专项基金项目(CXZX2017187)

Cloning and Expression Analysis of miR166 Primary Gene during the Early Stage of Somatic Embryogenesis of Longan

ZHANG Qinglin,SU Liyao,LI Xue,ZHANG Shuting,XU Xiaoping,CHEN Xiaohui,WANG Peiyu,LI Rong,ZHANG Zihao,CHEN Yukun,LAI Zhongxiong*,and LIN Yuling*   

  1. Institute of Horticultural Biotechnology,Fujian Agriculture and Forestry University,Fuzhou 350002,China
  • Online:2018-08-25 Published:2018-08-25

摘要: 为了解龙眼miR166初级体基因Pri-miR166 S53结构特点及其前体和成熟体在龙眼体胚发生早期的表达模式,采用SMARTTM RACE试剂盒和PCR扩增技术克隆龙眼体胚早期miR166基因的初级体(Pri-miR166 S53),确认其转录起始位点,并预测其含有的潜在ORF;利用龙眼基因组数据库提取其启动子序列,预测其含有的顺式作用元件;用实时荧光定量PCR对龙眼体胚发生早期及不同激素处理下的胚性愈伤组织中miR166基因的前体(Pre-miR166 S53)和成熟体(miR166a.2)表达模式进行分析。结果表明:获得长317 bp的Pri-miR166 S53基因初级体序列,对其进行翻译,得到一条长13个氨基酸序列的miPEP(MLCFVDALFLIST)。利用生物信息学软件分析Pri-miR166 S53基因的启动子序列发现,除了具有 TATA/CAAT-box外,还含有生长素、脱落酸、乙烯、水杨酸、茉莉酸甲酯及spl、HSE等特异作用元件。实时荧光定量PCR分析表明,在2,4-D调控的龙眼体胚发生早期过程中,从胚性松散型愈伤组织发育到球形胚的过程中,Pri-miR166 S53基因的前体pre-miR166 S53和成熟体miR166a.2都表现为下调趋势;而在无2,4-D调控的龙眼体胚发生早期中,pre-miR166 S53和miR166a.2表达模式不同。此外,pre-miR166 S53随ABA和乙烯处理浓度升高呈下调表达,而对不同浓度2,4-D处理无应答;miR166a.2随2,4-D、ABA处理浓度升高呈下调表达,而在乙烯处理下呈上调表达。上述研究结果提示miR166前体和成熟体在对外源激素应答的模式上并不呈简单线性相关,可能存在多层次、多方位的调控。

关键词: 龙眼, miR166初级体, 启动子, 实时荧光定量PCR, 表达特性

Abstract: In order to understand the structural characteristics of primary miR166 S53 gene (Pri-miR166 S53)and the expression pattern of its precursor and mature miRNA in the early stage of somatic embryogenesis(SE)in longan,SMARTTM RACE kit and PCR amplification were used to clone the pri-miR166 S53,to confirm its transcription start site and predict its potential ORF;longan genome database was used to extract its promoter sequence and predict cis-acting element;real-time fluorescence quantitative PCR was used to analyze the expression pattern of miR166 gene precursor(Pre-miR166 S53)and mature(miR166a.2)in longan early SE and in embryogenic callus under different hormone treatments. The results showed that the primary sequence of Pri-miR166 S53 gene was obtained with a length of 317 bp,encoded 13 amino acid sequence of miPEP(MLCFVDALFLIST). Analysis of promoter sequence of Pri-miR166 S53 gene using bioinformatics software revealed that in addition to TATA/CAAT-box,it also contains auxin,abscisic acid,ethylene,salicylic acid,methyl jasmonate,spl,HSE,and other specific elements. Real-time fluorescence quantitative PCR showed that pre-miR166 S53 and mature miR166a.2 showed a down trend in the early stage of 2,4-D-regulated longan SE during the process from friable-embryogenic callus(EC)into globular embryos;however,pre-miR166 S53 and miR166a.2 showed different expression patterns in the early stage of SE without 2,4-D regulation. In addition,pre-miR166 S53 was down-regulated in EC at different concentrations of ABA and ethylene treatment,but no response to 2,4-D;miR166a.2 was down-regulated in EC under different concentrations of 2,4-D,ABA,but up-regulated under ethylene treatment. The above results suggest that pre-miR166 S53 and miR166a.2 are not simple linear correlation patterns in the response to exogenous hormone,there may be multi-level,multi-directional regulation.

Key words: Dimocarpus longan, miR166 primary, promoter, real-time fluorescence quantitative PCR, expression characteristic

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