巨桉EgrDREB2A基因结构及表达特性分析

doi:10.11707/j.1001-7488.20150210

摘要/Abstract

摘要：

【目的】从低温条件下巨桉mRNA抑制消减杂交(SSH)文库中筛选得到一个受低温诱导的基因 EgrDREB2A (Eucgr. G03094),通过对其蛋白序列特征、亚细胞定位特点以及低温、ABA和盐胁迫逆境处理条件下基因表达方式的分析,讨论 EgrDREB2A 在巨桉非生物逆境响应中所发挥的作用。【方法】使用SMART,MatInspector和MEGA等生物信息学软件,分析 EgrDREB2A 的序列和编码蛋白特征,搜寻启动子上的重要顺式作用元件并构建其蛋白序列进化树; 亚细胞定位采用基因枪轰击洋葱表皮的方法; EgrDREB2A 组织表达特异性及低温、ABA和盐胁迫条件下的基因表达分析分别采用半定量和定量RT-PCR方法进行; 在4 ℃低温下随处理时间(0.5,2,6,12,24,48 h)的基因数字表达谱基础上,用WGCNA和Cytoscape软件对 EgrDREB2A 进行基因共表达分析。【结果】 EgrDREB2A 编码的蛋白序列含有1个典型的AP2结构域,且结构域内部包含YRG和RAYD 2个保守区,属于DREB2类基因,进化树构建结果表明该基因属于DREB2类基因的亚型Ⅰ。 EgrDREB2A 启动子序列上含有多个与植物逆境响应相关的顺式作用元件。该基因有明显的核定位区,亚细胞定位结果也表明其编码蛋白主要在核中表达。正常条件下,根、茎和叶中 EgrDREB2A 都有表达,但叶片中表达水平相对较高。在不同低温(0,2,4,6,8 ℃)下 EgrDREB2A 被强烈诱导,4 ℃低温下随处理时间(0.5,2,6,12,24,48 h)的延长,其表达量呈上升趋势,不同时间处理下与 EgrDREB2A 具有共表达关系的基因多属于参与植物对逆境响应的基因。ABA(100 μmol·L^-1)对 EgrDREB2A 的表达表现为先促进而后抑制,而NaCl(200 mmol·L^-1)的处理则表现为先抑制后促进。昼夜节律同样影响 EgrDREB2A 的表达,光照下基因表达升高,黑暗条件下基因表达降低。【结论】巨桉 EgrDREB2A 属于DREB2类基因,其表达受低温诱导,同时受ABA、盐和昼夜节律的影响。该基因启动子序列上启动子元件及与其共表达基因大多与植物逆境响应有关。这些结果表明 EgrDREB2A 可能在巨桉抵抗非生物逆境因子的过程中发挥比较重要的作用。

关键词: 巨桉, EgrDREB2A, 基因表达, 非生物胁迫, 昼夜节律

Abstract:

【Objective】 A gene, EgrDREB2A , was isolated from the mRNA suppression subtractive hybridization library of Eucalyptus grandis (Eucgr. G03094). Based on the analysis of structure, subcellular localization of EgrDREB2A protein and gene expression under different treatments of low temperature, ABA and salt, the roles of EgrDREB2A in the resistance to abiotic stresses of Eucalyptus grandis were discussed. 【Method】 SMART and MatInspector softwares were used to analyze the protein structure of EgrDREB2A and the cis-elements in promoter sequence of the gene. Phylogenetic tree of DREB proteins was constructed by MEGA software. Subcellular localization of EgrDREB2A was characterized with the method of introducing EgrDREB2A-GFP fused genes into onion epidermal cells via gene gun bombardment. And, gene expression analysis in different tissue, under treatments of low temperature, ABA, salt and circadian rhythm were carried out by semi-quantitative and quantitative RT-PCR method respectively. For the gene co-expression of EgrDREB2A under different time treatment at 4 ℃, WGCNA and Cytoscape softwares were used. 【Result】 EgrDREB2A was classed into DREB2 group because the protein it encodes containing one AP2 domain which including a YRG and a RAYD conserved regions. The phylogenetic tree based on homology comparison showed it belonging to subtypeⅠof DREB2 group. Several cis-elements related with plant stress response were found in the EgrDREB2A promoter sequence. Nuclear localization with DREB2 merged protein with GFP implied EgrDREB2A mainly located in the nucleus. qRT-PCR result of EgrDREB2A under 0 ℃, 2 ℃,4 ℃, 6 ℃ and 8 ℃ revealed it was induced. Time course (0.5, 2, 6, 12, 24, 48 h) treatments to E. grandis seedlings at 4 ℃ also increased its expression as the time delayed. The genes co-expressed with EgrDREB2A under different time treatment at 4 ℃ were mostly associated with plant response to abiotic stresses. EgrDREB2A expression increased at the first and then decreased under 100 μmol·L^-1 ABA treatment. However, under the salt stress (200 mmol·L^-1), it was inhibited firstly and then induced. Circadian rhythm also regulates the EgrDREB2A expression, and the transcription level of it was promoted under light and hampered in the darkness. 【Conclusion】 EgrDREB2A is a transcriptional factor which was classified into DREB2 group. The expression of it was induced by low temperature and ABA, salt treatments and circadian rhythm also changed its transcription level. Most of cis-elements found in the promoter of EgrDREB2A and genes co-expressed with it were all associated with plant stress response. All these results showed EgrDREB2A possibly played an important role in the process of resistance to aboitic stresses in E. grandis.

Key words: Eucalyptus grandis, EgrDREB2A, gene expression, abiotic stress, circadian rhythm

中图分类号:

S718.46

魏晓玲, 程龙军, 窦锦青, 徐凤华. 巨桉EgrDREB2A基因结构及表达特性分析[J]. 林业科学, 2015, 51(2): 80-89.

Wei Xiaoling, Cheng Longjun, Dou Jinqing, Xu Fenghua. The Structure and Expression Characteristics of EgrDREB2A Gene in Eucalyptus grandis[J]. Scientia Silvae Sinicae, 2015, 51(2): 80-89.

参考文献

王京京, 童再康, 黄程前, 等. 2012. 巨桉EgrCBF 1和EgrCBF2 基因的克隆和胁迫响应表达分析. 林业科学, 48(10): 41-48.
(Wang J J, Tong Z K, Huang C Q, et al. 2012. Cloning of EgrCBF 1 and EgrCBF2 genes from Eucalyptus grandis and their expression under stresses. Scientia Silvae Sinicae, 48(10):41-48.)
王亚红, 刘缙, 王玉国. 2010. 高质量提取银杏种仁总 RNA 的改良方法. 中国农学通报, 26(15): 48-52.
(Wang Y H, Liu J, Wang Y G. 2010. An improved method RNA isolation from seeds of Ginkgo biloba L. Chinese Agricultural Science Bulletin, 26(15):48-52.)
Agarwal P K, Agarwal P, Reddy M K, et al. 2006. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep, 25(12): 1263-1274.
Agarwal P, Agarwal P K, Nair S, et al. 2007. Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity. Mol Genet Genomics, 277(2): 189-198.
Avonce N, Leyman B, Mascorro-Gallardo J O, et al. 2004. The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signaling. Plant Physiol, 136(3): 3649-3659.
Bieniawska Z, Espinoza C, Schlereth A, et al. 2008. Disruption of the Arabidopsis circadian clock is responsible for extensive variation in the cold-responsive transcriptome. Plant Physiol, 147(1): 263-279.
Chinnusamy V, Schumaker K, Zhu J K. 2004. Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. Journal of experimental botany, 55(395): 225-236.
Dubouzet J G, Sakuma Y, Ito Y, et al. 2003. OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J, 33(4): 751-763.
Egawa C, Kobayashi F, Ishibashi M, et al. 2006. Differential regulation of transcript accumulation and alternative splicing of a DREB2 homolog under abiotic stress conditions in common wheat. Genes Genet Syst, 81(2): 77-91.
Fowler S G, Cook D, Thomashow M F. 2005. Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiol, 137(3): 961-968.
Herrera-Rodriguez M B, Perez-Vicente R, Maldonado J M. 2007. Expression of asparagine synthetase genes in sunflower (Helianthus annuus) under various environmental stresses. Plant Physiol Biochem, 45(1): 33-38.
Jaglo-Ottosen K R, Gilmour S J, Zarka D G, et al. 1998. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science, 280(5360): 104-106.
Kayal E W, Navarro M, Marque G, et al. 2006. Expression profile of CBF-like transcriptional factors genes from Eucalyptus in response to cold. Journal of Expression Botany, 57(10): 2455-2496.
Lim C J, Hwang J E, Chen H, et al. 2007. Over-expression of the Arabidopsis DRE/CRT-binding transcription factor DREB2C enhances thermotolerance. Biochem Biophys Res Commun, 362(2): 431-436.
Liu L, Zhu K, Yang Y, et al. 2008. Molecular cloning, expression profiling and trans-activation property studies of a DREB2-like gene from chrysanthemum (Dendranthema vestitum). J Plant Res, 121(2): 215-226.
Liu Q, Kasuga M, Sakuma Y, et al. 1998. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10(8): 1391-1406.
Matsukura S, Mizoi J, Yoshida T, et al. 2010. Comprehensive analysis of rice DREB 2 -type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes. Mol Genet Genomics, 283(2): 185-196.
Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K. 2012. AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta, 1819(2): 86-96.
Nakashima K, Ito Y, Yamaguchi-Shinozaki K. 2009. Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiology, 149(1): 88-95.
Nakashima K, Takasaki H, Mizoi J, et al. 2012. NAC transcription factors in plant abiotic stress responses. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1819(2): 97-103.
Novillo F, Medina J, Salinas J. 2007. Arabidopsis CBF1 and CBF3 have a different function than CBF2 in cold acclimation and define different gene classes in the CBF regulon. Proc Natl Acad Sci USA, 104(52): 21002-21007.
Okamuro J K, Caster B, Villarroel R, et al. 1997. The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc Natl Acad Sci USA, 94(13): 7076-7081.
Paul M J, Primavesi L F, Jhurreea D, et al. 2008. Trehalose metabolism and signaling. Annu Rev Plant Biol, 59: 417-441.
Puranik S, Sahu P P, Srivastava P S, et al. 2012. NAC proteins: regulation and role in stress tolerance. Trends in Plant Science, 17(6): 369-381.
Qin F, Sakuma Y, Tran L S, et al. 2008. Arabidopsis DREB2A-interacting proteins function as RING E3 ligases and negatively regulate plant drought stress-responsive gene expression. Plant Cell, 20(6): 1693-1707.
Sakuma Y, Liu Q, Dubouzet J G, et al. 2002. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochem Biophys Res Commun, 290(3): 998-1009.
Sakuma Y, Maruyama K, Osakabe Y, et al. 2006. Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression. Plant Cell, 18(5): 1292-1309.
Shen Y G, Zhang W K, He S J, et al. 2003. An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress. Theor Appl Genet, 106(5): 923-930.
Stockinger E J, Gilmour S J, Thomashow M F. 1997. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA, 94(3): 1035-1040.
Usadel B, Blasing O E, Gibon Y, et al. 2008. Multilevel genomic analysis of the response of transcripts, enzyme activities and metabolites in Arabidopsis rosettes to a progressive decrease of temperature in the non-freezing range. Plant Cell Environ, 31(4): 518-547.
Wang S, Wei X L, Cheng L J, et al. 2014. Identification of a C2H2-type zinc finger gene family from Eucalyptus grandis and its response to various abiotic stresses. Biologia Plantarum, 58(2): 385-390.
Wang H, Liu D, Sun J, et al. 2005. Asparagine synthetase gene TaASN 1 from wheat is up-regulated by salt stress, osmotic stress and ABA. J Plant Physiol, 162(1): 81-89.
Xu S P, Wei Z M. 1998. Introduction to method of microprojectile bombardment and its application. Plant Physiology Communications, 34(1):41-43.
Zhao J, Ren W, Zhi D, et al. 2007. Arabidopsis DREB1A/CBF3 bestowed transgenic tall fescue increased tolerance to drought stress. Plant Cell Rep, 26(9): 1521-1528.

[1]	沈乐, 徐建民, 李光友, 陆钊华, 杨雪艳, 朱映安, 胡杨, 宋佩宁, 郭文仲. 尾叶桉与巨桉杂种F₁代生长性状遗传分析[J]. 林业科学, 2019, 55(7): 68-76.
[2]	刘道凤, 王霞, 代银, 杨建峰, 马婧, 李名扬, 眭顺照. 蜡梅转录因子CpTAF10基因的克隆及功能分析[J]. 林业科学, 2019, 55(6): 176-183.
[3]	卢惠君, 李子义, 梁瀚予, 岳远志, 周天畅, 杨玉璋, 王玉成, 及晓宇. 刚毛柽柳NAC24基因的表达及抗逆功能分析[J]. 林业科学, 2019, 55(3): 54-63.
[4]	范伟健, 相伟芳, 王静, 白鹏华, 潘丽娜, 杨艺新, 朱耿平, 李敏. 农药与紫外胁迫对白蛾周氏啮小蜂小分子热激蛋白sHSP12.2基因表达的影响[J]. 林业科学, 2019, 55(2): 128-136.
[5]	张扬, 杜琳, 唐贤丰, 刘唤唤, 周功克, 柴国华. 杨树BAG基因的鉴定及表达模式分析[J]. 林业科学, 2019, 55(1): 138-145.
[6]	魏明科, 俞金健, 黄晓龙, 刘琼瑶, 黄华宏, 林二培, 童再康. 杉木NAC转录因子基因ClNAC1的克隆、表达及单核苷酸多态性分析[J]. 林业科学, 2018, 54(9): 49-59.
[7]	王娟, 陈辉. 华山松大小蠹冷休克结合蛋白的鉴定与表达[J]. 林业科学, 2018, 54(4): 58-66.
[8]	张鹤华, 刘嘉欣, 罗朝兵, 张凌云. 青杄转录因子PwERF8及其启动子序列的克隆与分析[J]. 林业科学, 2018, 54(3): 48-60.
[9]	马婧, 李政, 陈信立, 张升艳, 眭顺照, 李名扬. 蜡梅CpEXP1基因启动子的克隆及活性分析[J]. 林业科学, 2018, 54(3): 61-72.
[10]	李建波, 贾会霞, 张进, 刘伯斌, 胡建军, 王丽娟, 卢孟柱. 毛白杨PtoWOX4a基因过表达对次生生长的影响[J]. 林业科学, 2018, 54(2): 52-59.
[11]	刘长英, 张梦, 魏从进, 许亚珍, 曹博宁, 郑莎, 范伟, 向仲怀, 赵爱春. 转桑树MAPK5基因拟南芥在不同逆境胁迫下的抗性分析[J]. 林业科学, 2017, 53(9): 35-44.
[12]	孙化雨, 李利超, 赵韩生, 杨意宏, 王思宁, 高志民. 黄藤2个NAC基因的分子特征及其SSR分子标记开发[J]. 林业科学, 2017, 53(8): 132-140.
[13]	唐绯绯, 赵玉琳, 王培龙, 冯德明, 宋怡, 高彩球. 刚毛柽柳ThP5CR基因的克隆及抗逆功能分析[J]. 林业科学, 2017, 53(7): 1-9.
[14]	李楠, 郑勇奇, 丁红梅, 柳新红, 盛炜彤, 江波, 李海波. 低温胁迫下短枝木麻黄耐寒相关基因的差异表达分析[J]. 林业科学, 2017, 53(7): 62-71.
[15]	游韩莉, 袁义杭, 李长江, 张凌云. 青杄MYB转录因子基因PwMYB20的克隆及表达分析[J]. 林业科学, 2017, 53(5): 23-32.