Crops ›› 2024, Vol. 40 ›› Issue (2): 54-61.doi: 10.16035/j.issn.1001-7283.2024.02.007

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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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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

Table 1

Primers used in this study"

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

Fig.1

Amplified sequence of StCWIN1 gene promoter M: DL2000; 1: Amplified product of the promoter sequence."

Fig.2

Regulatory elements in StCWIN1 promoter"

Table 2

Analysis of regulatory elements in StCWIN1 promoter"

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

Fig.3

GUS staining in different tissues of transgenic PStCWIN1-GUS and wild type Arabidopsis a-e: Transgenic Arabidopsis (a: root; b: leaf; c: stem; d: flower; e: fruit pod); f-j: Wild type Arabidopsis (f: leaf; g: flower; h: fruit pod; i: root; j: stem)."

Fig.4

GUS activity in transgenic Arabidopsis with PStCWIN1-GUS (a) Enzymatic assay of GUS activity, (b) Quantification of GUS activity, ***: P < 0.001."

Fig.5

The relative expression of GUS gene in transgenic PStCWIN1-GUS Arabidopsis under drought stress “**”indicates significant differences between treatments (P < 0.01)."

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