渗透胁迫下玉米自然反义转录本cis-NATZmNAC48启动子的功能分析

doi:10.3724/SP.J.1006.2024.33013

摘要/Abstract

摘要：

前期研究发现自然反义转录本cis-NAT_ZmNAC48可负调控干旱响应基因ZmNAC48, 为了进一步探索cis- NAT_ZmNAC48的功能, 本研究以cis-NAT_ZmNAC48 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-NAT_ZmNAC48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-NAT_ZmNAC48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, Pro_cis_-NAT_ZmNAC48:GUS和Pro_ZmNAC48:GUS转基因拟南芥根、茎、叶中均有GUS表达, 且渗透胁迫处理后Pro_cis_-NAT_ZmNAC48: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Pro_ZmNAC48: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-NAT_ZmNAC48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-NAT_ZmNAC48启动子区域DNA甲基化情况, 发现在cis-NAT_ZmNAC48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-NAT_ZmNAC48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-NAT_ZmNAC48, acts as a negative regulator for maize drought stress response gene ZmNAC48. To further characterize the function of cis-NAT_ZmNAC48, we used cis-NAT_ZmNAC48 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-NAT_ZmNAC48 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-NAT_ZmNAC48 and ZmNAC48 promoters were constructed and transgenic Arabidopsis thaliana was obtained by infecting inflorescences. GUS staining analysis showed that Pro_cis_-NAT_ZmNAC48:GUS and Pro_ZmNAC48: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 Pro_{cis-NATZmNAC48}:GUS transgenic Arabidopsis were significantly decreased and increased significantly in Pro_ZmNAC48:GUS transgenic Arabidopsis, which indicating that both cis-NAT_ZmNAC48 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-NAT_ZmNAC48. 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-NAT_ZmNAC48 regulation.

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

毛燕, 郑名敏, 牟成香, 谢吴兵, 唐琦. 渗透胁迫下玉米自然反义转录本cis-NAT_ZmNAC48启动子的功能分析[J]. 作物学报, 2024, 50(2): 354-362.

MAO Yan, ZHENG Ming-Min, MOU Cheng-Xiang, XIE Wu-Bing, TANG Qi. Function analysis of the promoter of natural antisense transcript cis- NAT_ZmNAC48 in maize under osmotic stress[J]. Acta Agronomica Sinica, 2024, 50(2): 354-362.

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链接本文: https://zwxb.chinacrops.org/CN/10.3724/SP.J.1006.2024.33013

https://zwxb.chinacrops.org/CN/Y2024/V50/I2/354

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参考文献 23

[1]	Magali L, Patrice D, Gert T, Kathleen M, Yves M, Yves V D P, Pierre R, Stephane R. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res, 2002, 30: 325-327.
[2]	Higo K, Ugawa Y, Iwamoto M, Korenaga T. Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res, 1999, 27: 297-300.
[3]	Hrishikesh U, Lingaraj S, Sanjib K P. Molecular Physiology of Osmotic Stress in Plants. Berlin, Germany: Springer, 2013 [2023-06-05]. doi: 10.1007/978-81-322-0807-5.
[4]	Lapidot M, Pilpel Y. Genome-wide natural antisense transcription: coupling its regulation to its different regulatory mechanisms. EMBO Rep, 2006, 7: 1216-1222. pmid: 17139297
[5]	Borsani O, Zhu J, Verslues P E, Sunkar R, Zhu J K. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell, 2005, 123: 1279-1291.
[6]	Zhao X, Li J, Lian B, Gu H, Li Y, Qi Y. Global identification of Arabidopsis lncRNAs reveals the regulation of MAF4 by a natural antisense RNA. Nat Commun, 2018, 9: 5056. doi: 10.1038/s41467-018-07500-7
[7]	Fang J, Zhang F, Wang H, Wang W, Zhao F, Li Z, Sun C, Chen F, Xu F, Chang S, Wu L, Bu Q, Wang P, Xie J, Chen F, Huang Y, Zhang Y, Zhu X, Han B, Deng X, Chu C. Ef-cd locus shortens rice maturity duration without yield penalty. Proc Natl Acad Sci USA, 2019, 116: 18717-18722. doi: 10.1073/pnas.1815030116 pmid: 31451662
[8]	Fedaka H, Palusinskaa M, Krzycczmonika K, Brzezniak L, Yatusevich R, Pietras Z, Kaczanowski S, Swiezewski S. Control of seed dormancy in Arabidopsis by a cis-acting noncoding antisense transcript. Proc Natl Acad Sci USA, 2016, 113: E7846-E7855.
[9]	Fedaka H, Palusinskaa M, Krzyczmonika K, Brzezniaka L, Yatusevicha R, Pietrasa Z, Szymon K B, Szymon S. Antisense transcription represses Arabidopsis seed dormancy QTL DOG1 to regulate drought tolerance. EMBO Rep, 2017, 18: 2186-2196. doi: 10.15252/embr.201744862 pmid: 29030481
[10]	Zubko E, Merer P. A natural antisense transcript of the Petunia hybrida Sho gene suggests a role for an antisense mechanism in cytokinin regulation. Plant J, 2007, 52: 1131-1139. doi: 10.1111/tpj.2007.52.issue-6
[11]	Mehdi J D S, Cécile L, Christophe R, Qingya S, Yves P. A rice cis-natural antisense RNA acts as a translational enhancer for its cognate mRNA and contributes to phosphate homeostasis and plant fitness. Plant Cell, 2013, 25: 4166-4182. doi: 10.1105/tpc.113.116251
[12]	Mao Y, Xu J, Wang Q, Li G, Tang X, Liu T, Feng X, Wu F, Li M, Xie W B, Lu Y L. A natural antisense transcript acts as a negative regulator for the maize drought stress response gene ZmNAC48. J Exp Bot, 2021, 72: 2790-2806. doi: 10.1093/jxb/erab023
[13]	Clough S J, Bent A F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J, 1998, 16: 735-743. doi: 10.1046/j.1365-313x.1998.00343.x pmid: 10069079
[14]	Gruntman E, Qi Y, Slotkin R K, Roeder T, Martienssen R A, Sachidanandam R. Kismeth: analyzer of plant methylation states through bisulfite sequencing. BMC Bioinform, 2008, 9: 371. doi: 10.1186/1471-2105-9-371
[15]	Xu J, Wang Q, Freeling M, Zhang X, Xu Y, Mao Y, Tang X, Wu F K, Lan H, Cao M J, Rong T Z, Damon L, Lu Y L. Natural antisense transcripts are significantly involved in regulation of drought stress in maize. Nucleic Acids Res, 2017, 45: 5126-5141. doi: 10.1093/nar/gkx085 pmid: 28175341
[16]	Narusaka Y, Nakashima K, Shinwari Z K, Sakuma Y, Furihata T, Abe H, Narusaka M, Shinozaki K, Yamaguchi-Shinozaki K. Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses. Plant J, 2003, 34: 137-148. doi: 10.1046/j.1365-313X.2003.01708.x
[17]	Nakashima K, Fujita Y, Katsura K, Maruyama K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis. Plant Mol Biol, 2006, 60: 51-68. pmid: 16463099
[18]	Rushton P J, Somssich I E, Ringler P, Shen Q J. WRKY transcription factors. Trends Plant Sci, 2010, 15: 247-258. doi: 10.1016/j.tplants.2010.02.006 pmid: 20304701
[19]	Hiroshi A, Kazuko Y S, Takeshi U, Toshisuke I, Daijiro H, Kazuo S. Role of Arabidopsis MYC and MYB homologs in drought and abscisic acid-regulated gene expression. Plant Cell, 1997, 9: 1859-1868.
[20]	Krebs J E. 江松敏译. Lewin基因X (中文版). 北京: 科学出版社, 2013. pp 588-612.
	Krebs J E. Jiang S M, trans trans. Lewin Genes X. Beijing: Science Press, 2013. pp 588-612 (in Chinese).
[21]	Smith J, Sen S, Weeks R J, Eccles M R, Chatterjee A. Promoter DNA hypermethylation and paradoxical gene activation. Trends Cancer, 2020, 6: 392-406. doi: S2405-8033(20)30067-4 pmid: 32348735
[22]	Zhou Z, Liu C, Qin M, Li W, Hou J, Shi X, Dai Z, Yao W, Tian B, Lei Z, Li Y, Wu Z. Promoter DNA hypermethylation of TaGli-gamma-2.1 positively regulates gluten strength in bread wheat. J Adv Res, 2022, 36: 163-173. doi: 10.1016/j.jare.2021.06.021
[23]	Fei Y, Xue Y, Du P, Yang S, Deng X. Expression analysis and promoter methylation under osmotic and salinity stress of TaGAPC1 in wheat (Triticum aestivum L.). Protoplasma, 2017, 254: 987-996. doi: 10.1007/s00709-016-1008-5

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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-NAT_ZmNAC48启动子序列扩增 Amplification of the promoter in cis-NAT_ZmNAC48	TATGGTTAGGCCCACTACTAG	GTACGTGCCGGCTGGCCAC
PNS-1F/1R	ZmNAC48启动子序列扩增 Amplification of the promoter in ZmNAC48	ATGCAACCCTGGTTATGTGCT	TGTCCCCAAAGAAATTCCTTT
pCAMBIA1391-Cis-NAT_ZmNAC48	Cis-NAT_ZmNAC48启动子序列载体构建 Construction of cis-NAT_ZmNAC48promoter 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-NAT_ZmNAC48 promoter	ATTGGGGATATATAGAAATTTTGTATATAA	CAAAAAAACGACACTTGATAAATACCCTC
1F/1R	ZmNAC48启动子甲基化检测 Detected the methylation of ZmNAC48 promoter	GGATGAATTATTTATATTTAGTTTTT	ACCTACCTCCTACCAATTTAACACA

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

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