巨桉GRF基因家族生物信息学分析及其在不同氮水平下的组织表达模式

Bioinformatics analysis of GRF gene family in Eucalyptus grandis and their tissue expression patterns under different nitrogen levels

【目的】从全基因组水平上鉴定巨桉生长调节因子(growth-regulating factor,GRF)基因家族成员,分析其在不同氮素水平下的组织表达模式,为巨桉GRF基因功能研究及氮高效利用桉树品种的培育奠定基础。【方法】在NCBI网站中选择巨桉基因组,用拟南芥和毛果杨的GRF蛋白序列与巨桉基因组数据库进行BLAST蛋白序列同源比对,并通过InterPro和SMART数据库进行保守结构域分析,获得巨桉GRF基因。利用在线网站Ex-pasy protparam、SignalP-5.0、WoLF PSORT和SOPMA,对巨桉GRF蛋白进行基本理化性质分析、信号肽预测、亚细胞定位及二级结构预测。利用MEGA7.0软件构建系统进化树,使用MEME在线平台分析EgrGRF蛋白的保守结构域和基序,用Plant CARE预测基因上游的顺式作用元件,并使用TBtools软件将结果可视化。以2年生巨桉苗为供试材料,浇灌氮素浓度分别为45(高氮)、15(常规氮)和1.5(低氮)mmol/L的营养液,4 d后取样,采用实时荧光定量PCR技术检测EgrGRF基因在3种氮素水平下的叶片、茎和根中的表达情况。【结果】鉴定得到6个巨桉GRF基因家族成员(EgrGRF1~EgrGRF6),分布于4条染色体上;6个EgrGRF蛋白的氨基酸数目为296~605个,分子质量为33415.86~64630.89 u,理论等电点为7.29~8.85,脂溶指数为39.93~64.79,均为亲水性蛋白;无规则卷曲和α-螺旋为主要二级结构元件,均定位于细胞核上,未检测到信号肽存在。系统进化树分析表明,巨桉、毛果杨和拟南芥的GRF家族成员可分为4组,各组中EgrGRF蛋白数量分别为0,3,1和2个,多数EgrGRF成员与毛果杨亲缘关系较近。基因结构及蛋白基序分析结果显示,巨桉GRF基因家族成员具有2~4个内含子;6个EgrGRF蛋白均具有CX_(9)CX_(10)CX_(2)H和QX_(3)LX_(2)Q保守基序。顺式作用元件分析表明,EgrGRF基因启动子区含有茉莉酸甲酯、脱落酸、分生组织表达及低温等响应元件。实时荧光定量PCR结果显示,EgrGRF2和EgrGRF6基因在低氮处理的巨桉叶片中优势表达,EgrGRF2、EgrGRF3、EgrGRF5和EgrGRF6基因在低氮处理根中的表达量较高氮和常规氮处理显著升高,EgrGRF4基因在常规氮处理茎中的表达量最高。【结论】鉴定获得6个巨桉GRF基因家族成员,其中EgrGRF4基因可能参与茎生长发育的调控,EgrGRF2、EgrGRF3、EgrGRF5和EgrGRF6基因可能参与了巨桉对低氮胁迫的响应。

【Objective】The members of growth-regulating factor(GRF)gene family of Eucalyptus grandis were identified at the genome-wide level and their tissue expression patterns under different nitrogen levels were analyzed to provide foundation for studying GRF gene function and cultivating varieties with high nitrogen use efficiency.【Method】The Eucalyptus grandis genome was selected from the NCBI website.The GRF protein sequences of Arabidopsis thaliana and Populus trichocarpa were homologously aligned with the Eucalyptus grandis genome database,and the conserved domain analysis was performed by InterPro and SMART databases.The online websites Ex-pasy protparam,SignalP-5.0,WoLF PSORT and SOPMA were used to analyze the basic physicochemical properties,signal peptide prediction,subcellular localization and secondary structure prediction of GRF protein.The phylogenetic tree was constructed by MEGA7.0 software.The conserved domains and motifs of EgrGRF protein were analyzed by MEME online platform.The cis-acting elements in the gene upstream were predicted by Plant CARE,and the results were visualized by TBtools software.Two-year-old Eucalyptus grandis seedlings were selected for tests by nutrient solutions with nitrogen concentrations of 45(high nitrogen),15(normal nitrogen)and 1.5(low nitrogen)mmol/L,respectively.After 4 days,the expression of EgrGRF gene in leaves,stems and roots under different nitrogen levels was detected by real-time fluorescence quantitative PCR.【Result】Six GRF gene family members of EgrGRF1-EgrGRF6 were identified and distributed on four chromosomes.The number of amino acids of 6 EgrGRF proteins ranged from 296 to 605,the molecular weight ranged from 33415.86 to 64630.89 u,the theoretical isoelectric point ranged from 7.29 to 8.85,and the fat solubility index ranged from 39.93 to 64.79.All proteins were hydrophilic.Random coil andα-helix were the main secondary structural elements located in the nucleus,while no signal peptide was detected.Phylogenetic tree analysis showed that the GRF family members of Eucalyptus grandis,Populus trichocarpa and Arabidopsis thaliana could be divided into 4 groups with numbers of 0,3,1 and 2,respectively.Most EgrGRF members were closely related to Populus trichocarpa.The gene structure and protein motif analysis showed that the GRF gene family members of Eucalyptus grandis had 2-4 introns.The six EgrGRF proteins had CX_(9)CX_(10)CX_(2)H and QX_(3)LX_(2)Q conserved motifs.Analysis of cis-acting elements showed that the promoter region of EgrGRF gene contained methyl jasmonate,abscisic acid,meristem expression and low temperature response elements.Real-time quantitative PCR showed that EgrGRF2 and EgrGRF6 genes dominantly expressed in leaves of Eucalyptus grandis under low nitrogen treatment.The expression levels of EgrGRF2,EgrGRF3,EgrGRF5 and EgrGRF6 genes in roots under low nitrogen treatment were significantly higher than those in other treatments.The expression level of EgrGRF4 gene was the highest in stems under normal nitrogen treatment.【Conclusion】Six GRF gene family members were identified.EgrGRF4 gene may play a role in the regulation of stem growth and development,and EgrGRF2,EgrGRF3,EgrGRF5 and EgrGRF6 genes may be involved in the response of Eucalyptus grandis to low nitrogen stress.