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作物学报 ›› 2022, Vol. 48 ›› Issue (3): 590-596.doi: 10.3724/SP.J.1006.2022.14016

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析

周悦(), 赵志华, 张宏宁, 孔佑宾*()   

  1. 河北农业大学农学院/教育部华北作物种质资源研究与利用重点实验室, 河北保定 071001
  • 收稿日期:2021-01-26 接受日期:2021-06-16 出版日期:2022-03-12 网络出版日期:2021-07-24
  • 通讯作者: 孔佑宾
  • 作者简介:E-mail: 1584868048@qq.com
  • 基金资助:
    河北省自然科学基金项目(C2018204090);现代农业科技奖励性后补助资金项目(17927670H)

Cloning and functional analysis of the promoter of purple acid phosphatase gene GmPAP14 in soybean

ZHOU Yue(), ZHAO Zhi-Hua, ZHANG Hong-Ning, KONG You-Bin*()   

  1. North China Key Laboratory for Germplasm Resources of Education Ministry/College of Agronomy, Hebei Agricultural University, Baoding 071001, Hebei, China
  • Received:2021-01-26 Accepted:2021-06-16 Published:2022-03-12 Published online:2021-07-24
  • Contact: KONG You-Bin
  • Supported by:
    Natural Science Foundation of Hebei Province(C2018204090);Modern Agricultural Science and Technology Award Post-subsidy Fund Project(17927670H)

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摘要：

大豆紫色酸性磷酸酶基因GmPAP14受低磷诱导表达, 其超表达显著提高植物有机磷利用效率, 为进一步探究其调控机制, 本研究以GmPAP14 cDNA序列检索大豆参考基因组, 获取基因上游启动子序列, 设计引物克隆了中黄15 GmPAP14启动子序列。利用PLACE与PlantCARE预测启动子调控元件发现, 该序列中含有增强子调控元件、组织特异表达元件, 根特异表达元件、转录因子PHR1结合的PIBS元件等。构建了GmPAP14启动子3个5°端缺失片段融合GUS的植物表达载体PGmPAP14-2568-GUS、PGmPAP14-2238-GUS、PGmPAP14-1635-GUS, 并通过Floral dip法获得转基因拟南芥。利用GUS染色和活性测定分析GmPAP14启动子不同片段表达活性发现, 正常磷条件下各片段转基因拟南芥均在根尖表达, 低磷条件下GUS染色可扩展到成熟区和根毛, 另外转PGmPAP14-2238-GUS植株的GUS活性最高。这些结果为后续的基因调控研究奠定重要基础。

关键词: 大豆, GmPAP14, 启动子, 诱导表达, 低磷

Abstract:

The expression of GmPAP14 was induced under low phosphorus condition, and its overexpression could significantly improve the utilization efficiency of organic phosphorus in Arabidopsis. In order to further explore its regulatory mechanism, the promoter sequence of GmPAP14 was cloned according to the sequence of soybean reference genome. The regulatory elements of GmPAP14 promoter were predicted by the database PLACE and PlantCARE, which showed that it contained enhancer regulatory elements, tissue-specific expression elements, root-specific expression elements, and P1BS elements (transcription factor PHR1 binding sites). PGmPAP14-2568-GUS, PGmPAP14-2238-GUS, and PGmPAP14-1635-GUS were constructed and transferred into Arabidopsis thaliana via Floral dip method. The expressional activities of three fragments of GmPAP14 promoter were analyzed through GUS staining and activity measurement. The results revealed that GmPAP14 promoter was mainly expressed in root tip under Pi condition, and GUS staining was extended to the elongation area, mature area, and root hair after low phosphorus treatment. Additionally, Arabidopsis plants with PGmPAP14-2238-GUS had the highest activity among them. These results lay an important foundation for the further study of gene regulation.

Key words: soybean, GmPAP14, promoter, induced expression, low phosphorus

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 用途
Function
PGmPAP14-F/R F: AGTCGAAGATGGGGATGT
R: TTTGCTCAAAACCCGTG		 GmPAP14启动子克隆
Cloning of GmPAP14 promoter
PGmPAP14-2568-F/R F: AAGCTTAGTCGAAGATGGGGATGT
R: GGATCCTTTGCTCAAAACCCGTG		 PGmPAP14-2568克隆
Cloning of PGmPAP14-2568
PGmPAP14-2238-F/R F: AAGCTTTAACAACACAAGTTGTGAATTTC
R: GGATCCTTTGCTCAAAACCCGTG		 PGmPAP14-2238克隆
Cloning of PGmPAP14-2238
PGmPAP14-1635-F/R F: AAGCTTCTGAGGAAAGACTACAGTATAC
R: GGATCCTTTGCTCAAAACCCGTG		 PGmPAP14-1635克隆
Cloning of PGmPAP14-1635
PGmPAP14-Gus-F/R F: GGATCACTAGAATCACGGGTTTTG
R: ACACTTTTCCCGGCAATAACATAC		 转基因植株检测
Detection of transgenic plants

图1

GmPAP14启动子克隆 M: DNA分子量标准DL2000; 1: GmPAP14启动子扩增产物。"

表2

GmPAP14启动子顺式调控元件分析"

调控元件
Regulation element		 功能注释
Functional annotation		 数目
Number
W box 诱导应答元件 Induced response element 2
P1BS PHR1转录因子结合位点 PHR1-binding site 1
RHERPATEXPA7 根尖特异表达元件 Root hair-specific cis-elemen 2
OSE1ROOTNODULE 组织特异元件 Organ-specific element 5
OSE2ROOTNODULE 组织特异元件 Organ-specific element 8
ROOTMOTIFTAPOX1 组织特异元件 Organ-specific element 20
A/T rich element 增强子元件 Enhancer element 1
ABRELATERD1 脱落酸响应元件 Cis-acting element involved in the abscisic acid responsiveness 1
GAREAT 赤霉素响应元件 GA-responsive element 2
TGACG-motif 茉莉酸甲酯响应元件 Cis-acting regulatory element involved in MeJA response 2
CAAT-box 启动子和增强子区的一般顺式作用元件
Common cis-acting element in promoter and enhancer regions		 40
TATA-box 转录起始位点-30核心启动子元件Core promoter element around -30 of transcription start 26

图2

GmPAP14启动子5°端缺失片段扩增 A: 5°缺失片段扩增。B: 5°缺失片段示意图。M: DNA分子量标准DL5000; 1: 含酶切位点Hind III/BamH I PGmPAP14-2568扩增; 2: 含酶切位点Hind III/BamH I PGmPAP14-2238扩增; 3: 含酶切位点Hind III/BamH I PGmPAP14-1635扩增。B: 5°缺失片段示意图。"

图3

转GmPAP14启动子T3拟南芥PCR检测 M: DNA分子量标准DL2000; 1: 水对照; 2: 野生型对照; 3: 质粒对照; 4~6: 转PGmPAP14-2568-GUS拟南芥; 7~9: 转PGmPAP14-2238-GUS拟南芥; 10~12: 转PGmPAP14-1635-GUS拟南芥。"

图4

转PGmPAP14-GUS拟南芥不同组织化学染色 A: 根; B: 叶片; C: 茎; D: 花。"

图5

不同磷条件下转GmPAP14启动子5°端缺失片段拟南芥GUS染色分析 PGmPAP14-2568-GUS、PGmPAP14-2238-GUS、PGmPAP14-1635-GUS分别代表转5°缺失拟南芥; +Pi: 磷酸二氢钾; +Po: 植酸磷。"

图6

不同磷条件下转GmPAP14启动子5°端缺失片段拟南芥GUS活性测定 PGmPAP14-2568-GUS、PGmPAP14-2238-GUS、PGmPAP14-1635-GUS分别代表转5°缺失拟南芥; +Pi: 磷酸二氢钾; +Po: 植酸磷。不同小写字母代表在0.05水平差异显著。"

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