摘要
【目的】通过对巨桉非生物逆境响应相关基因EgrZFP6(Eucgr.A01232)蛋白结构和基因功能的初步研究,探讨该基因在巨桉非生物逆境响应中所发挥的作用,为桉树抗逆育种提供理论基础。【方法】首先利用CDD在线软件分析EgrZFP6编码蛋白序列的结构域,并利用NCBI中的Blast软件搜索与EgrZFP6蛋白序列相似程度较高的其他物种中ZFP蛋白,用Clustalx进行多序列比对,联合分析、比较它们的结构域。然后,构建EgrZFP6∷s GFP融合载体,采用基因枪轰击洋葱表皮方法对EgrZFP6蛋白表达进行亚细胞定位;同时,构建35S∷EgrZFP6超表达载体,采用花序侵染法进行拟南芥遗传转化;对获得的超表达拟南芥转基因纯合株系,分析其正常条件、低温、干旱和高盐等非生物逆境处理下的表型变化;利用酵母双杂交法筛选到与EgrZFP6互作蛋白Egr ERF4(Eucgr.F01164),并对低温、干旱和高盐等非生物胁迫下巨桉植株中Egr ERF4的表达情况用实时荧光定量RT-PCR方法进行分析。【结果】巨桉EgrZFP6编码蛋白为1个典型C2H2型锌指结构蛋白,有2个包含QALGGH序列的植物特有锌指结构域,1个乙烯响应元件结合因子相关双性抑制子EAR基序和1个L-box基序;亚细胞定位结果表明EgrZFP6表达蛋白定位在细胞核中;与野生型对照相比,EgrZFP6超表达的拟南芥转化植株中,主根伸长生长受到一定抑制,对低温敏感性增强,PEG(1 g·L-1以上)处理能促进侧根增加和伸长,植株根伸长对高盐抑制作用的耐受性有一定程度提高。乙烯响应相关转录因子基因Egr ERF4编码蛋白能够与EgrZFP6编码蛋白互作;正常巨桉植株不同低温(-8,-4,0,4℃)2 h处理下,除-8℃外,Egr ERF4表达均呈现被诱导趋势;4℃低温不同时间(0.5,2,6,12,24,48 h)处理下,基因也被诱导表达;干旱条件下,随处理时间延长,基因表达被严重抑制,而高盐(200 mmol·L-1)胁迫则能促进Egr ERF4表达。【结论】EgrZFP6转录因子可能
【Objective】EgrZFP6（ Eucgr. A01232） is a gene which is involved in abiotic stresses response in Eucalyptus grandis. With the study of protein structure and function of EgrZFP6,the roles it possibly played in abiotic stress response in E. grandis were discussed in order to provide a basis for stress resistance breeding of Eucalyptus. 【Method 】 The EgrZFP6 protein structure was analyzed with CDD online software. And,sequences of ZFP protein in other plant species that have high similarity with EgrZFP6 were downloaded from NCBI after Blast software was used. Multiple alignments for these sequences were finished with Clustalx and their motifs were analyzed. EgrZFP6∷s GFP fused expression vector was also constructed and transformed into onion epidermal cells via gene gun bombardment to identify subcellular localization of EgrZFP6. Transformation of 35 S∷EgrZFP6 into Arabidopsis thaliana was via floral-dip method. Two homozygous lines of EgrZFP6 over-expression in A. thaliana were obtained,their phenotype under normal condition,low temperature,drought and high salinity treatments were evaluated compared to wild type（ COL）. Based on Yeast 2 Hybridization,Egr ERF4（ Eucgr. F01164）,a protein can interact with EgrZFP6,was screened from the Yeast 2 Hybridization library. The expression of Egr ERF4 under low temperature,drought and salinity in E. grandis seedlings was also analyzed. Protein thatcan interact with EgrZFP6 were screened and verified by Yeast 2 Hybridization. Gene expression of Egr ERF4 under abiotic stresses was analyzed by real time fluorescence quantitative RT-PCR method.【Result】 The EgrZFP6 is a classic C2 H2 type zinc finger protein. There are 2 zinc finger domains with QALGGH sequence which is specific for plants in it. An ERF（ ethylene responsive element binding factor） associated amphiphilic repression（ EAR） motif and a L-box motif were also found in the protein sequence. Result of subcellular localization revealed the protein EgrZFP6 encoded was localized in the nuclear
作者
王晓荣
程龙军
徐凤华
倪晓祥
陆军
Wang Xiaorong;Cheng Longjun;Xu Fenghua;Ni Xiaoxiang;Lu Jun(State Key Laboratory of Subtropical Silviculture Zhefiang A＆F University Hangzhou 311300)
出处
《林业科学》
EI
CAS
CSCD
北大核心
2017年第11期60-68,共9页
Scientia Silvae Sinicae
基金
国家自然科学基金项目"巨桉锌指结构蛋白基因EgrZPCT在抗冷胁迫中功能和调控机制研究"(31270657)
浙江省科技厅林木新品种选育重大科技专项"沿海防护林重点树种高抗品种选育"(2016C02056-9)
