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作物学报 ›› 2024, Vol. 50 ›› Issue (2): 354-362.doi: 10.3724/SP.J.1006.2024.33013

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

渗透胁迫下玉米自然反义转录本cis-NATZmNAC48启动子的功能分析

毛燕1,*(), 郑名敏1, 牟成香1, 谢吴兵2, 唐琦2   

  1. 1成都师范学院, 四川成都 611130
    2四川农业大学玉米研究所, 四川成都 611130
  • 收稿日期:2023-03-05 接受日期:2023-09-13 出版日期:2024-02-12 网络出版日期:2023-09-27
  • 通讯作者: *毛燕, E-mail: 2469018993@qq.com
  • 作者简介:E-mail: 2469018993@qq.com
  • 基金资助:
    四川省自然科学基金项目(2022NSFSC0152)

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 Published:2024-02-12 Published online:2023-09-27
  • Contact: *E-mail: 2469018993@qq.com
  • Supported by:
    General Project of Natural Science Foundation of Sichuan Province(2022NSFSC0152)

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摘要：

前期研究发现自然反义转录本cis-NATZmNAC48可负调控干旱响应基因ZmNAC48, 为了进一步探索cis- NATZmNAC48的功能, 本研究以cis-NATZmNAC48 cDNA序列、ZmNAC48蛋白质编码序列检索玉米B73参考基因组, 获取基因上游启动子序列, 并利用PlantCARE[1]和New PLACE[2]预测启动子调控元件, 发现cis-NATZmNAC48和ZmNAC48启动子序列中除含有CAAT-box, TATA-box等基本元件外, 还含有激素响应元件以及转录因子结合元件等。构建cis-NATZmNAC48和ZmNAC48启动子融合GUS的表达载体, 并通过花序侵染法获得转基因拟南芥。分析GUS染色和GUS酶活性发现, Procis-NATZmNAC48:GUS和ProZmNAC48:GUS转基因拟南芥根、茎、叶中均有GUS表达, 且渗透胁迫处理后Procis-NATZmNAC48:GUS转基因拟南芥中GUS 基因的表达量和GUS酶活性显著降低, 而ProZmNAC48:GUS转基因拟南芥中GUS基因的表达量和GUS酶活性显著增加, 可见cis-NATZmNAC48和ZmNAC48启动子均响应渗透胁迫。DNA甲基化是影响启动子活性的调控事件之一, 本研究通过分析cis-NATZmNAC48启动子区域DNA甲基化情况, 发现在cis-NATZmNAC48序列前400~1000 bp存在DNA甲基化修饰, 渗透胁迫处理后, 该甲基化区域甲基化富集情况发生了显著的变化, 但发生显著性变化的甲基化位点并未在顺式调控元件上。这些结果为后续cis-NATZmNAC48的调控分析奠定了重要的基础。

关键词: 玉米, 自然反义转录本, 启动子, 渗透胁迫

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

表1

本研究所用引物"

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

表2

Cis-NATZmNAC48 启动子中主要顺式调控元件"

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

表3

ZmNAC48启动子中顺式调控元件"

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

图1

Cis-NATZmNAC48和ZmNAC48启动子功能分析 A~C: ProZmNAC48:GUS和Procis-NATZmNAC48:GUS转基因拟南芥中GUS染色, GUS表达量及GUS酶活。CK表示将14 d大小的拟南芥放于1/2 MS液体培养基中生长6 h; 20% PEG-6000表示将14 d大小的拟南芥放于含20% PEG-6000 1/2 MS液体培养基中生长6 h。WT表示野生型拟南芥哥伦比亚0型。显著性分析采用t检验, **表示P < 0.01。"

图2

Cis-NATZmNAC48和ZmNAC48启动子区域DNA甲基化 A: cis-NATZmNAC48启动子区域DNA甲基化。B: ZmNAC48启动子区域DNA甲基化。CK表示玉米材料正常的营养液中生长的玉米材料, 20% PEG-6000表示在含有20% PEG-6000营养液中处理24 h。"

图3

Cis-NATZmNAC48和ZmNAC48启动子区域DNA甲基化分析 A~E: 用BSP检测玉米耐旱自交系AC7643和干旱敏感自交系AC7729/TZSRWB中cis-NATZmNAC48启动子区域DNA甲基化情况。F: 用BSP检测ZmNAC48启动子区域DNA甲基化情况。CK表示对照, 20% PEG-6000表示用20% PEG-6000处理玉米材料6 h。红色线条表示甲基化类型GC, 绿色线条表示甲基化类型CHG, 蓝色线条表示甲基化类型CHH。显著性分析采用卡方检验, **表示P < 0.01。图A~B, D~F中所用到的单克隆个数分别为: 50、44、29、29和11。"

图4

Cis-NATZmNAC48 启动子甲基化区域顺式元件预测 红色箭头所指位置为胁迫处理后DNA甲基化发生显著性变化的位点。图中前面4个箭头所在位置是渗透胁迫处理后, 玉米自交系AC7643中检测到的甲基化富集发生显著变化的位点, 最后1个箭头所在位置是AC7729/TZSRWB中检测到的甲基化富集发生显著变化的位点。"

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