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作物杂志,2017, 第2期: 51–58 doi: 10.16035/j.issn.1001-7283.2017.02.009

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

大豆GmWRI1a基因启动子克隆及序列分析

闫丽,杨强,邵宇鹏,李丹丹,王志坤,李文滨   

  1. 东北农业大学/大豆生物学教育部重点实验室/农业部东北大豆生物学与遗传育种重点实验室,150030,黑龙江哈尔滨
  • 收稿日期:2016-11-21 修回日期:2017-02-26 出版日期:2017-04-15 发布日期:2018-08-26
  • 通讯作者: 王志坤,李文滨
  • 作者简介:闫丽,硕士研究生,研究方向为大豆遗传育种与分子生物学
  • 基金资助:
    营养功能型转基因大豆新品种培育(2016ZX08004-003);2016年东北农业大学“学术骨干”项目

Cloning and Sequence Analysis of GmWRI1a Gene Promoter in Soybean

Yan Li,Yang Qiang,Shao Yupeng,Li Dandan,Wang Zhikun,Li Wenbin   

  1. Northeast Agricultural University/Key Laboratory of Soybean Biology, Chinese Ministry of Education/Key Laboratory of Soybean Biology and Genetics Breeding,Chinese Agriculture Ministry,Harbin 150030,Heilongjiang,China
  • Received:2016-11-21 Revised:2017-02-26 Online:2017-04-15 Published:2018-08-26
  • Contact: Zhikun Wang,Wenbin Li

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摘要：

以大豆基因组文库Phytozome公布的大豆Williams82基因组序列为参考,应用Primer Premier 5.0软件设计引物,用PCR技术扩增了大豆GmWRI1a基因的启动子序列,构建了重组克隆载体pGM-T-pGmWRI1a,并通过PCR扩增对阳性克隆进行鉴定送测序。克隆获得GmWRI1a基因启动子序列1 686bp,该启动子序列除含有必需的起始转录位点、TATA-box、CTTA-box外还包含多个顺式作用元件,如光应答元件、赤霉素应答元件、表达分生组织相关元件、抗旱诱导元件等。同时,构建了该启动子植物表达载体pBI-pGmWRI1a,通过PCR扩增、限制性酶切对阳性克隆进行了鉴定,为启动子的功能研究奠定基础。大豆GmWRI1a基因启动子克隆与序列分析,将为进一步研究大豆GmWRI1a基因的表达调控及其功能分析提供参考。

关键词: GmWRI1a基因启动子, 大豆, 序列分析, 顺式调控元件

Abstract:

According to the Williams 82’s genomic sequences reported in the Phytozome, primers were designed by Primer Premier 5.0. The promoter product sequence of GmWRI1a gene was amplified by PCR method. The recombinant vectors pGM-T-pGmWRI1a and PBI121-pGmWRI1a were constructed. The positive clones were identified by PCR amplification and restriction enzyme digestion. The full length of the GmWRI1a promoter was 1 687bp. The promoter sequence contained the necessary initiation transcription sites, such as TATA-box,CTTA-box and multiple other cis-acting elements: light responsiveness element, gibberellin response element, cis-acting regulatory element related to meristem expression, drought-inducing elements, and others. Cloning and characterization of the GmWRI1a gene promoter will provide basis for the further study of regulation and functional analysis of GmWRI1a gene in soybean.

Key words: Promoter of GmWRI1a gene, Soybean, Sequence analysis, Cis-regulatory element

图1

东农47 DNA提取结果 M：核酸分子量标准DL15000 Nucleic acid molecular weight standard 15000;1-4：DNA提取液DNA extracts"

图2

GmWRI1a基因启动子PCR扩增结果 M：核酸分子量标准DL2000,下同 Nucleic acid molecular weight standard 2000, the same below;1-3：GmWRI1a基因启动子GmWRI1a gene promoter（1 686bp）"

图3

重组质粒pGM-T-pGmWRI1a的PCR鉴定 1-4：pGM-T-pGmWRI1a重组质粒pGM-T-pGmWRI1a recombinant plasmid;5：空白对照 Blank control"

图4

GmWRI1a基因启动子序列比对结果"

图5

GmWRI1a基因启动子序列分析 序列中可能的顺式作用元件用方块标出;预测的转录起始位点用黑体表示;下画线ATG为GmWRI1a基因的起始密码子"

表1

GmWRI1a基因启动子顺式调控元件的位置与推测功能"

调控元件Regulatory sequence 位置Position 核心序列Core sequence 预测功能Function
ACE
 797（+）
 ACGTGGA
 光应答元件
Cis-acting element involved in light responsiveness
AE-Box

 176（+）,
1278（+）,
655（–）		 AGAAAC

 光响应模块
Part of a module for light response
ATC-motif
 1227（–）
 AGTAATCT
 光应答元件
Part of a conserved DNA module involved in light responsiveness
CATT-motif
 210（+）
 GCATTC
 光响应一部分
Part of a light responsive
GA-motif
 835（–）
 AAAGATGA
 光响应元件
Part of a light responsive element
G-box
 795（+）,
796（–）		 TGACGTGG
CACGTC		 光反应调节元件
Cis-acting regulatory element involved in light responsiveness
调控元件Regulatory sequence 位置Position 核心序列Core sequence 预测功能Function
GT1-motif

 674（+）,
1056（+）,
914（–）		 GGTTAA

 光反应元件
Light responsive element
CTA-box
 1008（+）
 GCCACT
 表达分生组织相关
Cis-acting regulatory element related to meristem expression
P-Box
 896（–）
 CCTTTTG
 赤霉素应答元件
Gibberellin-responsive element
GARE-motif
 603（+）,
1482（+）		 AAACAGA
TCTGTTG		 赤霉素响应元件
Gibberellin-responsive element
CE3
 802（+）
 GACGCGTGT
 ABA和VP1响应元件
Cis-acting element involved in ABA and VP1 responsiveness
MBS
 791（+）
 TAACTG
 MYB参与抗旱诱导结合位点
MYB binding site involved in drought-inducibility
Skn-1_motif
 362（+）,
664（+）		 GTCAT
 胚乳表达所需的调节元件
Cis-acting regulatory element required for endosperm expression
GCN4_motif

 370（+）,
1005（+）,
818（+）		 TGAGTCA

 胚乳表达顺式调控元件
Cis-regulatory element involved in endosperm expression

图6

重组质粒pBI-pGmWRI1a的PCR鉴定 1-3：pGM-T-pGmWRI1a重组质粒 pGM-T-pGmWRI1a recombinant plasmid;4：空白对照 Blank control"

图7

重组质粒pBI-pGmWRI1a的Hind Ⅲ、XbaⅠ酶切鉴定 1：pBI-pGmWRI1a重组质粒pBI-pGmWRI1a recombinant plasmid"

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