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作物杂志,2024, 第2期: 54–61 doi: 10.16035/j.issn.1001-7283.2024.02.007

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

马铃薯细胞壁蔗糖转化酶基因StCWIN1启动子克隆与表达及其在干旱胁迫下的作用分析

张玉1(), 杨文静1, 刘璇1, 聂峰杰1, 张丽1, 石磊1, 张国辉2, 郭志乾2, 巩檑1()   

  1. 1宁夏农林科学院农业生物技术研究中心/宁夏农业生物技术重点实验室，750002，宁夏银川
    2宁夏农林科学院固原分院，756000，宁夏固原
  • 收稿日期:2023-02-21 修回日期:2023-03-05 出版日期:2024-04-15 发布日期:2024-04-15
  • 通讯作者: 巩檑，主要从事马铃薯资源鉴定及分子育种研究，E-mail：tea_gl@126.com
  • 作者简介:张玉，研究方向为植物生物技术育种，E-mail：nxzy_work@163.com
  • 基金资助:
    国家自然科学基金(31860419);国家自然科学基金(32260504);宁夏回族自治区农业育种专项(2019NYYZ01-2);宁夏自然科学基金(2021AAC05015)

Cloning and Expression Analysis of Potato StCWIN1 Gene Promoter and Its Role under Drought Stress

Zhang Yu1(), Yang Wenjing1, Liu Xuan1, Nie Fengjie1, Zhang Li1, Shi Lei1, Zhang Guohui2, Guo Zhiqian2, Gong Lei1()   

  1. 1Research Center of Agricultural Biotechnology, Ningxia Academy of Agriculture and Forestry Sciences /Ningxia Key Laboratory of Agricultural Biotechnology, Yinchuan 750002, Ningxia, China
    2Guyuan Branch of Ningxia Academy of Agriculture and Forestry Sciences, Guyuan 756000, Ningxia, China
  • Received:2023-02-21 Revised:2023-03-05 Online:2024-04-15 Published:2024-04-15

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摘要：

植物细胞壁蔗糖转化酶（cell wall invertase，CWIN）是源、库组织蔗糖代谢及胁迫应答的关键酶。本研究利用基因步移法克隆马铃薯StCWIN1启动子片段，应用PlantCARE在线软件对启动子区域的作用元件进行分析，将融合StCWIN1启动子与GUS报告基因的表达载体转化拟南芥野生型，并利用组织化学染色和GUS实时定量PCR技术探究启动子表达活性、组织表达特性和响应干旱胁迫的表达规律。结果表明，克隆获得StCWIN1基因上游1956 bp启动子序列，其中包含核心调控、植物激素、防御及胁迫、光响应等关键元件；StCWIN1启动子在根、柱头和果荚组织中的表达活性高于其他组织；转StCWIN1启动子拟南芥株系叶片中GUS表达量高于野生型，且干旱胁迫显著抑制了GUS相对表达量。本研究克隆得到具有活性的StCWIN1启动子，基于研究结果推测目的基因可能参与根、花和果实等器官发育，对干旱胁迫也发挥应答调节作用。

关键词: 马铃薯, 细胞壁蔗糖转化酶, 启动子, GUS活性, 干旱胁迫, 表达分析

Abstract:

Cell wall invertase (CWIN) is a key enzyme for stress responses and sucrose metabolism in source and sink tissues. In this study, the promoter sequence of StCWIN1 was cloned by genomic walking, and its cis-regulatory elements were analysed by using PlantCARE. A GUS-fused StCWIN1 promoter expression vector was constructed and subsequently transformed into Arabidopsis thaliana. Promoter activity, tissue expression characteristics and responsiveness to drought stress were investigated by histochemical staining and real-time quantitative PCR. The results showed that the StCWIN1 promoter was 1956 bp long and contained enhancer- regulated, phytohormone-responsive, protective/stress-responsive and light-responsive elements. The expression activity of StCWIN1 promoter were higher in roots, stigmas, and pods than other tissues. The expression of GUS in leaves of Arbidopsis Thaliana with StCWIN1 promoter was higher than that of wild type, and the value was significantly reduced under drought stress. The identification of the active StCWIN1 promoter in the present study suggests that StCWIN1 may be involved in the development of organs such as roots, flowers and fruit pods, and may also function in response to drought stress.

Key words: Solanum tuberosum L., Cell wall invertase, Promoter, GUS activity, Drought stress, Expression analysis

表1

本试验所用引物

引物名称Primer name 引物序列Primer sequence (5’-3’) 用途Function
pCWIN-NeGW
 F1：AACATTTTTCACATCAACCGTG
F2：GATGAAACAGTTCCACACACTACC		 启动子扩增
pCWIN-positive selection
 F：CAAATGATAAGTTACACCATC
R：TTTAAATCAGATTTCTACGAG		 阳性植株筛选
GUS-RT-F/R
 F：ATACCGAAAGGTTGGGCAGG
R：CGGCAATAACATACGGCGTG		 实时定量GUS基因
Actin
 F：GGTAACATTGTGCTCAGTGGTGG
R：AACGACCTTAATCTTCATGCTGC		 实时定量内参基因

图1

StCWIN1基因启动子序列扩增 M：DL2000；1：启动子序列扩增产物。

图2

StCWIN1启动子调控元件

表2

StCWIN1启动子调控元件分析

元件Element 数量Amount 位置Position 序列Sequence 功能Function
ERE

 3

 993(+)
1018(-)
1016(+)		 ATTTTAAA

 乙烯响应元件

Box 4

 3

 737(+)
1559(-)
1098(-)		 ATTAAT

 光响应元件

ABRE
 2
 812(+)
1259(+)		 ACGTG
 脱落酸响应元件
O2-site
 2
 749(-)
1672(-)		 GATGATGTGG/GTTGACGTGA
 参与玉米蛋白代谢调控元件
CGTCA-motif 1 1606(+) CGTCA 茉莉酸甲酯响应元件
TGA-element 1 78(+) AACGAC 生长素响应元件
LTR 1 604(+) CCGAAA 低温胁迫响应元件
TC-rich repeats 1 1286(-) GTTTTCTTAC 防御及胁迫响应元件
ARE 1 1369(+) AAACCA 厌氧诱导必需的调控元件
MYB 1 503(+) TAACCA MYB转录因子作用元件
circadian 1 179(-) CAAAGATATC 昼夜节律调节元件

图3

转PStCWIN1-GUS拟南芥与野生型拟南芥不同组织中的GUS染色 a~e：转基因拟南芥（a：根；b：叶片；c：茎；d：花；e：果荚）；f~j：野生型拟南芥（f：叶片；g：花；h：果荚；i：根；j：茎）。

图4

转PStCWIN1-GUS拟南芥GUS活性 (a) GUS活性分析，(b) GUS定量分析，***：P < 0.001。

图5

干旱胁迫下转PStCWIN1-GUS拟南芥GUS相对表达量 “**”表示处理间差异显著（P < 0.01）。

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