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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (2): 354-362.doi: 10.3724/SP.J.1006.2024.33013

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Function analysis of the promoter of natural antisense transcript cis- NATZmNAC48 in maize under osmotic stress

MAO Yan1,*(), ZHENG Ming-Min1, MOU Cheng-Xiang1, XIE Wu-Bing2, TANG Qi2   

  1. 1Chengdu Normal University, Chengdu 611130, Sichuan, China
    2Maize Research Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
  • Received:2023-03-05 Accepted:2023-09-13 Online:2024-02-12 Published:2023-09-27
  • Contact: *E-mail: 2469018993@qq.com E-mail:2469018993@qq.com
  • Supported by:
    General Project of Natural Science Foundation of Sichuan Province(2022NSFSC0152)

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Abstract:

Previous studies indicate that natural antisense transcript, cis-NATZmNAC48, acts as a negative regulator for maize drought stress response gene ZmNAC48. To further characterize the function of cis-NATZmNAC48, we used cis-NATZmNAC48 cDNA sequence and ZmNAC48 protein coding sequence to retrieve maize B73 reference genome and obtain the upstream promoter. PlantCARE and New PLACE were used to predict promoter regulatory elements, which revealed that the promoter of cis-NATZmNAC48 and ZmNAC48 contained not only CAAT-box, TATA-box, and other basic elements, but also hormone response elements and transcription factor binding element. Plant expression vectors of GUS fusion with cis-NATZmNAC48 and ZmNAC48 promoters were constructed and transgenic Arabidopsis thaliana was obtained by infecting inflorescences. GUS staining analysis showed that Procis-NATZmNAC48:GUS and ProZmNAC48:GUS was expressed in roots, stems, and leaves of Arabidopsis thaliana. After osmotic stress treatment, the relative expression level of GUS gene and GUS enzyme activity of Procis-NATZmNAC48:GUS transgenic Arabidopsis were significantly decreased and increased significantly in ProZmNAC48:GUS transgenic Arabidopsis, which indicating that both cis-NATZmNAC48 and ZmNAC48 promoters responded to osmotic stress. DNA methylation was one of the regulatory events that affected promoter activity. In this study, we found that DNA methylation modification existed in the promoter region of cis-NATZmNAC48. After osmotic stress treatment, the methylation enrichment changed significantly, but the methylation sites with significant changes were not in the cis-regulatory elements. These results laid an important basis for the analysis of cis-NATZmNAC48 regulation.

Key words: maize, natural antisense transcript, promoter, osmotic stress

Table 1

Primers used in this study"

引物名称
Primer name		 用途
Function		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')
PNA-1F/1R Cis-NATZmNAC48启动子序列扩增
Amplification of the promoter in
cis-NATZmNAC48		 TATGGTTAGGCCCACTACTAG GTACGTGCCGGCTGGCCAC
PNS-1F/1R ZmNAC48启动子序列扩增
Amplification of the promoter in ZmNAC48		 ATGCAACCCTGGTTATGTGCT TGTCCCCAAAGAAATTCCTTT
pCAMBIA1391-Cis-NATZmNAC48 Cis-NATZmNAC48启动子序列载体构建
Construction of cis-NATZmNAC48 promoter
vector		 tgggcccggcgcgccaagcttTATGGTTAGGCCCACTACTAG TCTTAGAATTCCCGGGGATCCGTACGTGCCGGCTGGCCAC
pCAMBIA1391- ZmNAC48 ZmNAC48启动子序列载体构建
Construction of ZmNAC48 promoter vector		 tgggcccggcgcgccaagcttATGCAACCCTGGTTATGTGCT TCTTAGAATTCCCGGGGATCCTGTCCCCAAAGAAATTCCTTT
3F/4R Cis-NATZmNAC48启动子甲基化检测
Detected the methylation of cis-NATZmNAC48
promoter		 ATTGGGGATATATAGAAATTTTGTATATAA CAAAAAAACGACACTTGATAAATACCCTC
1F/1R ZmNAC48启动子甲基化检测
Detected the methylation of ZmNAC48
promoter		 GGATGAATTATTTATATTTAGTTTTT ACCTACCTCCTACCAATTTAACACA

Table 2

Cis-elements of cis-NATZmNAC48 promoter"

调控元件
Regulatory elements		 数量
Number		 功能
Function
TATA-box 8 转录起始位点-30核心启动子元件 Core promoter element around -30 of transcription start
CAAT-box 9 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
A-box 1 顺式作用调控元件 Cis-acting regulatory element
Box 4 1 光响应元件 Part of a conserved DNA module involved in light responsivenes
ACE 1 光响应元件 Cis-acting element involved in light responsiveness
G-box 3 光响应元件 Cis-acting regulatory element involved in light responsiveness
TGACG-motif 3 茉莉酸甲酯响应元件 Cis-acting regulatory element involved in the MeJA-responsiveness
TGA-element 1 生长素响应元件 Auxin-responsive element
CGTCA-motif 1 茉莉酸甲酯响应元件 Cis-acting regulatory element involved in the MeJA-responsiveness
ABRE 8 脱落酸响应元件 Cis-acting element involved in the abscisic acid responsiveness
W box 1 WRKY转录因子结合位点 Binding site of WRKY
CCGTCC motif 2 诱导应答元件
These elements appear to be necessary but not sufficient for elicitor-or light-mediated PAL gene activation
AAGAA-motif 2
Unnamed__1 1
Unnamed__4 4
STRE 1

Table 3

The cis-elements of ZmNAC48 promoter"

调控元件
Regulatory elements		 数量
Number		 功能
Function
TATA-box 10 转录起始位点-30核心启动子元件 Core promoter element around -30 of transcription start
CAAT-box 8 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
3-AF3 binding site 1 保守DNA序列 Part of a conserved DNA module array (CMA3)
O2-site 1 玉米醇溶蛋白代谢 Cis-acting regulatory element involved in zein metabolism regulation
NON-box 1 分生组织特异表达 Cis-acting regulatory element related to meristem specific activation
GA-motif 1 光响应元件 Part of a light responsive element
TCCC-motif 1 光响应元件 Part of a light responsive element
ARE 1 厌氧诱导元件 Cis-acting regulatory element essential for the anaerobic induction
TGACG-motif 4 茉莉酸甲酯响应元件 Cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif 1 茉莉酸甲酯响应元件 Cis-acting regulatory element involved in the MeJA-responsiveness
MBS 1 MYB结合位点涉及干旱诱导 MYB binding site involved in drought-inducibility
Myb-binding site 6 MYB结合位点 MYB binding site
Myc 1
Unnamed__4 6
STRE 2

Fig. 1

Function analysis of cis-NATZmNAC48 and ZmNAC48 promoter A-C: GUS staining, GUS expression and GUS enzyme activity in transgenic Arabidopsis with ProZmNAC48:GUS and Procis-NATZmNAC48:GUS. CK represents 14-day Arabidopsis was grown in 1/2 MS liquid medium for 6 h, and 20% PEG-6000 represents 14-day Arabidopsis was grown in 1/2 MS liquid medium contained 20% PEG-6000 for 6 h, WT represents COL. ** indicates significant difference at P < 0.01 by Student’s t-test."

Fig. 2

DNA methylation in cis-NATZmNAC48 and ZmNAC48 promoter A: DNA methylation in cis-NATZmNAC48 promoter. B: DNA methylation in ZmNAC48 promoter. CK: maize grown in normal nutrient solution, and 20% PEG-6000 represents treated maize in containing 20% PEG-6000 nutrient solution for 24 h."

Fig. 3

DNA methylation characteristics in cis-NATZmNAC48 and ZmNAC48 promoter A-E: DNA methylation characteristics in cis-NATZmNAC48 promoter by bisulfite sequenced analysis in AC7643 and AC7729/TZSRWB. F: DNA methylation characteristics in cis-NATZmNAC48 promoter by bisulfite sequenced analysis. CK represents control, and 20% PEG-6000 represents treating maize material with 20% PEG-6000 for 6 h. Histograms indicated the percentages of CG (red), CHG (green), and CHH (blue), respectively. ** indicates significant difference at P < 0.01 by Chi-square test. The number of monoclones used in A-B, D-F is 50, 44, 29, 29, and 11, respectively."

Fig. 4

Cis-element prediction of cis-NATZmNAC48 promoter Red arrows indicate the sites where DNA methylation changes significantly under osmotic stress. Under the osmotic stress treatment, the sites of significant methylation enrichment were detected in AC7643, and the sites of significant methylation enrichment in AC7729/TZSRWB were detected in the last arrow."

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