巨桉EgrDREB2A基因结构及表达特性分析

The Structure and Expression Characteristics of Egr DREB2A Gene in Eucalyptus grandis

【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Egr DREB2A(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Egr DREB2A在巨桉非生物逆境响应中所发挥的作用。【方法】使用SMART,Mat Inspector和MEGA等生物信息学软件,分析Egr DREB2A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;Egr DREB2A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Egr DREB2A进行基因共表达分析。【结果】Egr DREB2A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。Egr DREB2A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Egr DREB2A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Egr DREB2A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Egr DREB2A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(100μmol·L-1)对Egr DREB2A的表达表现为先促进而后抑制,而Na Cl(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Egr DREB2A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Egr DREB2A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Egr DREB2A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。

[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＆nbsp;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.