玉米ZmGRAS13基因的克隆及功能研究

doi:10.3724/SP.J.1006.2024.33060

摘要/Abstract

摘要：

GRAS家族是植物特有的一类转录因子，在调控植物生长发育和响应逆境胁迫等方面发挥着重要作用。探究玉米(Zea mays L.) GRAS家族基因功能将为玉米新种质创制提供重要的基因资源。本研究克隆获得了ZmGRAS13基因(Zm00001eb401210)，利用生物信息学分析、实时荧光定量PCR (qPCR)等技术对该基因的基本特性、组织表达特性及逆境胁迫下表达模式等进行分析。生物信息学分析结果显示，该基因编码序列全长为1638 bp，编码545个氨基酸；ZmGRAS13蛋白不具有跨膜结构，分子量为60.79 kD，理论等电点为5.86，具有GRAS家族所特有的保守结构域。对基因启动子上游2 kb序列进行分析，发现该序列含有与逆境胁迫、激素响应及光响应等相关的顺式作用元件。qPCR分析表明，ZmGRAS13基因在玉米不同组织中均有表达，且茎中的表达量最高；同时该基因在不同非生物胁迫处理条件下均有不同程度的诱导表达。玉米原生质体瞬时表达实验表明，ZmGRAS13蛋白定位于细胞核。在分别含有不同浓度梯度的NaCl、甘露醇(mannitol)、脱落酸(abscisic acid, ABA)、茉莉酸(jasmonic acid, JA)和水杨酸(salicylic acid, SA)的1/2 MS固体培养基上，转ZmGRAS13基因拟南芥株系的根长均显著长于野生型拟南芥；在土壤中，高盐和干旱处理下转基因拟南芥株系较野生型拟南芥生长状态更好，且绿叶率高于野生型。转ZmGRAS13基因拟南芥与野生型相比，抗逆生理指标MDA含量降低、叶绿素含量增加、以及POD和CAT活性增强，且差异均显著。由此推测，ZmGRAS13基因可能参与玉米生长发育调控和对逆境胁迫应答及激素信号转导途径。本研究为进一步解析ZmGRAS13在玉米中的生物学功能提供了重要的参考依据。

关键词: 玉米, ZmGRAS13, 转录因子, 渗透胁迫, 盐胁迫, 异源表达

Abstract:

GRAS family is a plant-specific transcription factor, which plays an important role in regulating plant growth and development and responding to stresses. Exploring the function of GRAS family genes in maize (Zea mays L.) provides the important genetic resources for the creation of new maize germplasm. In this study, ZmGRAS13 gene (Zm00001eb401210) was cloned, and its basic characteristics, tissue expression characteristics, and the relative expression patterns under stresses were analyzed by bioinformatics and qRT-PCR. Bioinformatics showed that the full-length coding sequence of this gene was 1638 bp, encoding 545 amino acids. ZmGRAS13 protein had no transmembrane structure, and the molecular weight of 60.79 kD, the theoretical isoelectric point of 5.86, and had a conserved domain unique to the GRAS family. The analysis of 2 kb sequence upstream of the gene promoter indicated that the sequence contained cis-acting elements related to stresses, hormone response, and light response. The qRT-PCR analysis showed that ZmGRAS13 gene was expressed in different tissues of maize, and the relative expression level in stem was the highest. At the same time, the gene has different degrees of induced expression under different abiotic stress treatment conditions. The transient expression experiment of maize protoplasts demonstrated that ZmGRAS13 protein was localized in the nucleus. On 1/2 MS solid medium containing different concentrations of NaCl, mannitol, abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA), respectively, the root length of ZmGRAS13 transgenic Arabidopsis lines was significantly longer than the control. In the soil, transgenic Arabidopsis lines grew better than the control under high salt and drought treatments, and the green leaf rate was higher than the control. Compared with the wild type, the content of stress resistance physiological index MDA decreased, the chlorophyll content increased, and the activities of POD and CAT increased in the transgenic ZmGRAS13 Arabidopsis thaliana, and the difference was significant difference. In conclusion, ZmGRAS13 gene may be involved in the regulation of maize growth and development, response to stresses and hormone signal transduction pathway. This study provides an important reference for the further analysis of the biological function of ZmGRAS13 in maize.

Key words: maize, ZmGRAS13, transcription factors, osmotic stress, salt stress, heterologous expression

折萌, 郑登俞, 柯照, 吴忠义, 邹华文, 张中保. 玉米ZmGRAS13基因的克隆及功能研究[J]. 作物学报, 0, (): 0-.

SHE Meng, ZHENG Deng-Yu, KE Zhao, WU Zhong-Yi, ZOU Hua-Wen, ZHANG Zhong-Bao. Cloning and functional analysis of ZmGRAS13 gene in maize[J]. Acta Agronomica Sinica, 0, (): 0-.

参考文献

[1] Khan Y, Xiong Z, Zhang H, Liu S, Yaseen T, Hui T. Expression and roles of GRAS gene family in plant growth, signal transduction, biotic and abiotic stress resistance and symbiosis formation-a review. Plant Biol, 2022, 24: 404–416.

[2] Bolle C. The role of GRAS proteins in plant signal transduction and development. Planta, 2004, 218: 683–692.

[3] Guiltinan M J, Miller L. Molecular characterization of the DNA-binding and dimerization domains of the bZIP transcription factor, EmBP-1. Plant Mol Biol, 1994, 26: 1041–1053.

[4] Heery D M, Kalkhoven E, Hoare S, Parker M G. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature, 1997, 387: 733–736.

[5] Guo Y, Wu H, Li X, Li Q, Zhao X, Duan X, An Y, Lyu W, An H. Identification and expression of GRAS family genes in maize (Zea mays L.). PLoS One, 2017, 12: e0185418.

[6] 张文霞, 刁志娟, 吴为人. 植物GRAS蛋白研究的新进展. 分子植物育种, 2016, 14: 1159–1165.

Zhang W X, Diao Z J, Wu W R. New advances in the study of plant GRAS proteins. Mol Plant Breed, 2016, 14: 1159–1165 (in Chinese with English abstract).

[7] Tian C, Wan P, Sun S, Li J, Chen M. Genome-wide analysis of the GRAS gene family in rice and Arabidopsis. Plant Mol Biol, 2004, 54: 519–532.

[8] Heo J O, Chang K S, Kim I A, Lee M H, Lee S A, Song S K, Lee M M, Lim J. Funneling of gibberellin signaling by the GRAS transcription regulator scarecrow-like 3 in the Arabidopsis root. Proc NatI Acad Sci USA, 2011, 108: 2166–2171.

[9] Tanabe S, Onodera H, Hara N, Ishii-Minami N, Day B, Fujisawa Y, Hagio T, Toki S, Shibuya N, Nishizawa Y, Minami E. The elicitor-responsive gene for a GRAS family protein, CIGR2, suppresses cell death in rice inoculated with rice blast fungus via activation of a heat shock transcription factor, OsHsf23. Biosci Biotech Bioch, 2016, 80: 145–151.

[10] Mayrose M, Ekengren S K, Melech-Bonfil S, Martin G B, Sessa G. A novel link between tomato GRAS genes, plant disease resistance and mechanical stress response. Mol Plant Pathol, 2006, 7: 593–604.

[11] 周莲洁, 杨中敏, 张富春, 王艳. 新疆盐穗木GRAS转录因子基因克隆及表达分析. 西北植物学报, 2013, 33: 1091–1097.

Zhou L J, Yang Z M, Zhang F Q, Wang Y. Expression analysis and cloning of GRAS transcription factor gene from Halostachys capsica. Acta Bot Boreal-Occident Sin, 2013, 33: 1091–1097 (in Chinese with English abstract).

[12] 郭鹏, 邢新, 金华, 董燕. 玉米ZmSCL7的克隆及功能研究. 中国农业科学, 2013, 46: 2584–2591.

Guo P, Xing X, Jin H, Dong Y. Cloning and functional study of ZmSCL7 in Zea mays. Sci Agric Sin, 2013, 46: 2584–2591 (in Chinese with English abstract).

[13] Achard P, Cheng H, De Grauwe L, Decat J, Schoutteten H, Moritz T, Van Der Straeten D, Peng J, Harberd N P. Integration of Plant Responses to Environmentally Activated Phytohormonal Signals. Science, 2006, 311: 91–94.

[14] Huang W, Xian Z, Kang X, Tang N, Li Z. Genome-wide identification, phylogeny and expression analysis of GRAS gene family in tomato. BMC Plant Biol, 2015, 15: 209.

[15] Achard P, Gong F, Cheminant S, Alioua M, Hedden P, Genschik P. The cold-inducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism. Plant Cell, 2008, 20: 2117–2129.

[16] 郭华军. 拟南芥转录因子GRAS家族SCL15基因对干旱胁迫的响应分析. 西北农林科技大学硕士学位论文, 陕西杨凌, 2011.

Guo H J. Analysis of Arabidopsis GRAS Family Gene SCL15 Responded to Drought Stress. MS Thesis of Northwest A&F University, Yangling, Shaanxi, China, 2011 (in Chinese with English abstract).

[17] 丁雪峰, 刘鸿艳, 罗利军. 水稻OsGRAS1启动子的克隆及多样性分析. 上海农业学报, 2010, 26(4): 8–14.

Ding X F, Liu H Y, Luo L J. Cloning and diversity analysis of OsGRAS1 promoter in rice. Acta Agric Shanghai, 2010, 26(4): 8–14 (in Chinese with English abstract).

[18] 廖文彬, 彭明. 木薯赤霉素途径DELLA蛋白基因克隆及其对干旱胁迫的响应. 热带生物学报, 2012, 3(4): 298–304.

Liao W B, Peng M. Gene cloning of DELLA protein from cassava and its expression patterns under drought stress. J Trop Biol, 2012, 3(4): 298–304 (in Chinese with English abstract).

[19] Weng Y, Chen X, Hao Z, Lu L, Wu X, Zhang J, Wu J, Shi J, Chen J. Genome-wide analysis of the GRAS gene family in Liriodendron chinense reveals the putative function in abiotic stress and plant development. Front Plant Sci, 2023, 14: 1211853.

[20] Battaglia M, Rípodas C, Clúa J, Baudin M, Aguilar O M, Niebel A, Zanetti M E, Blanco F A. A nuclear factor Y interacting protein of the GRAS family is required for nodule organogenesis, infection thread progression, and lateral root growth. Plant Physiol, 2014, 164: 1430–1442.

[21] Gobbato E, Marsh J F, Vernié T, Wang E, Maillet F, Kim J, Miller J B, Sun J, Bano S A, Ratet P, Mysore K S, Dénarié J, Schultze M, Oldroyd G E. A GRAS-type transcription factor with a specific function in mycorrhizal signaling. Curr Biol, 2012, 22: 2236–2241.

[22] Bolle C, Koncz C, Chua N H. PAT1, a new member of the GRAS family, is involved in phytochrome A signal transduction. Gene Dev, 2000, 14: 1269–1278.

[23] Torres-Galea P, Hirtreiter B, Bolle C. Two GRAS proteins, SCARECROW-LIKE21 and PHYTOCHROME A SIGNAL TRANSDUCTION1, function cooperatively in phytochrome A Signal transduction. Plant Physiol, 2013, 161: 291–304.

[24] Fode B, Siemsen T, Thurow C, Weigel R, Gatz C. The Arabidopsis GRAS protein SCL14 interacts with class II TGA transcription factors and is essential for the activation of stress-inducible promoters. Plant Cell, 2008, 20: 3122–3135.

[25] Gao M J, Li X, Huang J, Gropp G M, Gjetvaj B, Lindsay D L, Wei S, Coutu C, Chen Z, Wan X C, Hannoufa A, Lydiate D J, Gruber M Y, Chen Z J, Hegedus D D. SCARECROW-LIKE15 interacts with HISTONE DEACETYLASE19 and is essential for repressing the seed maturation programme. Nat Commun, 2015, 6: 7243.

[26] Tong H, Jin Y, Liu W, Li F, Fang J, Yin Y, Qian Q, Zhu L, Chu C. DWARF AND LOW-TILLERING, a new member of the GRAS family, plays positive roles in brassinosteroid signaling in rice. Plant J, 2009, 58: 803–816.

[27] Li X, Qian Q, Fu Z, Xiong G, Zeng D, Wang X, Liu X, Teng S, Hiroshi F, Yuan M, Luo D, Han B, Li J. Control of tillering in rice. Nature, 2003, 422: 618–621.

[28] Meng F, Zheng X, Wang J, Qiu T, Yang Q, Fang K, Bhadauria V, Peng Y L, Zhao W. The GRAS protein OsDLA involves in brassinosteroid signalling and positively regulates blast resistance by forming a module with GSK2 and OsWRKY53 in rice. Plant Biotechnol J, 2023.

[29] Morohashi K, Minami M, Takase H, Hotta Y, Hiratsuka K. Isolation and characterization of a novel GRAS gene that regulates meiosis-associated gene expression. J Biol Chem, 2003, 278: 20865–20873.

[30] Li M, Sun B, Xie F, Gong R, Luo Y, Zhang F, Yan Z, Tang H. Identification of the GRAS gene family in the Brassica juncea genome provides insight into its role in stem swelling in stem mustard. Peer J, 2019, 7: e6682.

[31] Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Prot, 2007, 2: 1565–1572.

[32] Clough S J, Bent A F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J, 1998, 16: 735–743.

[33] Xu K, Chen S, Li T, Ma X, Liang X, Ding X, Liu H, Luo L. OsGRAS23, a rice GRAS transcription factor gene, is involved in drought stress response through regulating expression of stress-responsive genes. BMC Plant Biol, 2015, 15: 141.

[34] 王晓冬. 玉米GRAS转录因子基因ZmSCL14功能分析. 山东农业大学硕士学位论文, 山东泰安, 2020.

Wang X D. Functional analysis of maize GRAS transcription factor ZmSCL14 gene. MS Thesis of Shandong Agricultural University, Tai’an, Shandong, China, 2020 (in Chinese with English abstract).

[35] 殷龙飞, 王朝阳, 吴忠义, 张中保, 于荣. 玉米ZmGRAS31基因的克隆及功能研究. 作物学报, 2019, 45: 1029–1037.

Yin L F, Wang C Y, Wu Z Y, Zhang Z B, Yu R. Cloning and functional analysis of ZmGRAS31 gene in maize. Acta Agron Sin, 2019, 45: 1029–1037 (in Chinese with English abstract).

[36] Czikkel B E, Maxwell D P. NtGRAS1, a novel stress-induced member of the GRAS family in tobacco, localizes to the nucleus. J Plant Physiol, 2007, 164: 1220–1230.

[37] Ma H S, Liang D, Shuai P, Xia X L, Yin W L. The salt-and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance in Arabidopsis thaliana. J Exp Bot, 2010, 61: 4011–4019.

[38] Wang T T, Yu T F, Fu J D, Su H G, Chen J, Zhou Y B, Chen M, Guo J, Ma Y Z, Wei W L, Xu Z S. Genome-wide analysis of the GRAS gene family and functional identification of GmGRAS37 in drought and salt tolerance. Front Plant Sci, 2020, 11: 604690.

[39] Yuan Y, Fang L, Karungo S K, Zhang L, Gao Y, Li S, Xin H. Overexpression of VaPAT1, a GRAS transcription factor from Vitis amurensis, confers abiotic stress tolerance in Arabidopsis. Plant Cell Rep, 2016, 35: 655–666.

[40] Zhang S, Li X, Fan S, Zhou L, Wang Y. Overexpression of HcSCL13, a Halostachys caspica GRAS transcription factor, enhances plant growth and salt stress tolerance in transgenic Arabidopsis. Plant Physiol Bioch, 2020, 151: 243–254.

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