[Objective]NAC(NAM,ATAF and CUC2 proteins) is a family of special transcriptional factors in plants. The members in this family play important roles in life process of plants such as growth,development,signal transduction of hormones and stress responses. In order to provide more clues for further studies of the functions of EgrNAC1( Eucgr. I00058) gene,which possibly plays a very important role in Eucalyptus grandis,EgrNAC protein sequence,subcellular localization,gene expression under low temperature,high temperature,drought,salinity,ABA and MeJA were analyzed.[Method] Firstly,sequences of gene,promoter,and protein of EgrNAC1 were downloaded from genome database of Eucalyptus grandis. Then,bioinformatics software of SMART,MatInspector,and MEGA were applied to analyze the coding protein structure and cis-elements in promoter of EgrNAC1. EgrNAC1∷sGFP fused expression vector was also constructed with enzyme cutting method in the primary vector of pCAMBIA1300. And,the plasmid of EgrNAC1∷sGFP was transformed into onion epidermal cells via gene gun bombardment to identify subcellular localization of EgrNAC1 protein. Secondly,with the DGE ( digital gene expression) data from the treatment of different time course under 4 ℃with 3-month-old seedlings,genes co-expression with EgrNAC1 were analyzed with WGCNA software. Finally,to get more information of expression pattern of EgrNAC1 under different abiotic stress conditions,expression of EgrNAC1 under different temperatures ( - 8, - 4,0,4,8 ℃ ),time courses(0,2,6,12,24,48 h)at 4 ℃ ,high temperature (42 ℃ ), drought,salinity,ABA and MeJA were evaluated by real time fluorescence quantitative RT-PCR method .[Result] The NAC domain in EgrNAC1 contains 5 classical sub-domains A,B,C,D and E,including 2 α-helix and 5 β-sheets. There was also one nuclear localization sequence but no transmembrane region was found in the protein sequence. Phylogenetic analysis result showed that EgrNAC1 was classified into ATAF group in subfamily Ⅰ of NAC. In the NACs that were involved in stress responses,it belongs to SNAC-A subclass. ABRE,MBS,MCS,DREB and other cis-elements were found in the promoter of EgrNAC1 and most of them are related with abiotic stresses. Result of EgrNAC1 merged protein with GFP in onion cells showed the EgrNAC1 mainly expressed in the nuclear. In EgrNAC1 co-expression analysis under treatment of different time courses ( 0,2,6,12,24,48 h) at 4 ℃ ,among the 20 genes with the highest correlation coefficients,most were involved in the stress responses. EgrNAC1 was induced under the low temperature. The expression level of it increased with the time course under 4 ℃ treatment. For different low temperature treatments,the improvement of EgrNAC1 expression was higher under 4,8 ℃ compared to the other low temperature. The induction of EgrNAC1 were also found under treatment of drought,high salinity (200 mmol·L - 1 NaCl),ABA (100 μmol·L - 1 ) and MeJA ( 100μmol·L - 1 ). After 1-day treatment of drought stress,EgrNAC1 expression level increased,followed by a slow decrease. It only needs 2 hours to stimulate EgrNAC1 expression under MeJA treatment,but for the induction by ABA,it needs 24 hours.[Conclusion] EgrNAC1 is an important NAC gene which is not only involved in responses to abiotic stresses such as low temperature,drought,high salinity,but also possibly have interactions with ABA and MeJA hormone signal transduction in these stress responses.%[目的]NAC(NAM,ATAF和CUC2蛋白)是植物中一类特异转录因子,广泛参与植物生长、发育、激素信号转导和逆境响应过程.本文通过对巨桉EgrNAC1(Eucgr.I00058)编码蛋白结构、蛋白亚细胞定位特点,以及低温、干旱和高盐等非生物逆境条件下的基因表达分析,研究EgrNAC1基因与非生物逆境响应的关系,为其功能的深入研究提供基础.[方法]首先从巨桉基因组数据库下载EgrNAC1基因、启动子和编码蛋白序列,利用SMART、MatInspector和MEGA等软件对EgrNAC1蛋白结构特征、进化分类及启动子上的顺式作用元件进行分析;并以pCAMBIA1300为基础载体,用酶切法构建EgrNAC1∷sGFP载体,采用基因枪轰击洋葱表皮方法,对EgrNAC1蛋白表达的亚细胞定位特点进行研究.然后,以4℃ 不同时间处理的数字表达谱数据为基础,利用WGCNA软件进行基因共表达分析,了解低温下与EgrNAC1高度相关的共表达基因特征.为进一步了解EgrNAC1在不同非生物逆境处理下的表达特征,同样利用3个月巨桉无性系幼苗进行不同低温(-8,-4,0,4,8℃)、4℃ 不同时间(2,6,12,24,48 h)、高温(42℃)、高盐、干旱、脱落酸(ABA)和茉莉酸甲酯(MeJA)的不同处理,用实时荧光定量RT-PCR方法对这些处理下EgrNAC1的表达情况进行分析.[结果]EgrNAC1中NAC结构域包含A,B,C,D,E 5个典型亚结构域,其中含2个 α 螺旋5个 β 折叠片结构,有核定位序列,无跨膜域.进化树构建结果表明,EgrNAC1属于NAC家族的Ⅰ类亚家族ATAF子组,逆境类NAC分类中属于SNAC-A亚类.EgrNAC1启动子序列上含有ABRE、MBS、MCS和DREB等大量与逆境胁迫相关的顺式作用元件.亚细胞定位结果表明EgrNAC1在核中表达.4℃ 不同处理时间(0,2,6,12,24,48 h)下与EgrNAC1共表达相关系数最高的20个基因中,大多数基因参与逆境胁迫响应相关的调控过程.不同时间处理下,叶片中EgrNAC1诱导水平随处理时间延长不断提高;不同低温下,4℃ 和8℃ 处理中EgrNAC1的诱导水平相对较高;干旱处理1天后基因表达即受到诱导,然后又有所下降;盐(200 mmol·L-1 NaCl)胁迫48 h,才能诱导EgrNAC1表达;100μmol·L-1 ABA和100μmol·L-1 MeJA均能诱导叶片中EgrNAC1基因表达,MeJA诱导速度更快,处理2 h后基因表达即明显提高,而ABA的诱导效应则需要24 h.[结论]EgrNAC1是参与巨桉逆境胁迫响应的1个NAC类转录因子基因,其不仅参与低温、干旱和高盐的非生物逆境胁迫响应,还可能与ABA、MeJA信号转导有交叉互作效应.
展开▼
