甘蔗ScWRKY4基因的克隆与表达特性分析  

Cloning and Expression Characteristic Analysis of ScWRKY4 Gene in Sugarcane

作　　者：王玲[1] 刘峰[1] 戴明剑 孙婷婷[1] 苏炜华[1] 王春风 张旭[1] 毛花英 苏亚春[1,2] 阙友雄[1,2] WANG Ling;LIU Feng;DAI Ming-Jian;SUN Ting-Ting;SU Wei-Hua;WANG Chun-Feng;ZHANG Xu;MAO Hua-Ying;SU Ya-Chun;QUE You-Xiong(Key Laboratory of Sugarcane Biology and Genetic Breeding(Fujian),Ministry of Agriculture/Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China;Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization,Ministry of Education/Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China)

机构地区：[1]福建农林大学/农业部福建甘蔗生物学与遗传育种重点实验室,福建福州350002 [2]福建农林大学/教育部作物遗传育种与综合利用重点实验室,福建福州350002

出　　处：《作物学报》2018年第9期1367-1379,共13页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31671752和31101196);福建省杰出青年基金项目(2015J06006);福建省高校杰出青年科研人才计划项目(苏亚春-2017);国家现代农业产业技术体系建设专项(CARS-17)资助~~

摘　　要：WRKY是植物中特有的转录因子家族之一,在植物对生物和非生物逆境胁迫的应答反应中起重要调控作用。本研究基于课题组前期构建的甘蔗(Saccharum spp.)转录组数据库,从新台糖22(ROC22)中成功克隆到1个WRKY基因,命名为Sc WRKY4(Gen Bank登录号为MG852087)。序列分析发现,Sc WRKY4基因c DNA全长1265 bp,包含1个741 bp的完整开放读码框,编码246个氨基酸,该蛋白具有1个WRKYGQK保守结构域和C2H2锌指结构域,属于IIc类WRKY转录因子。生物信息学预测分析发现,Sc WRKY4蛋白为碱性的不稳定亲水性蛋白,不存在信号肽和跨膜结构域,蛋白二级结构元件缺少β螺旋。在本氏烟(Nicotiana benthamiana)叶片瞬时表达中,Sc WRKY4蛋白定位于细胞核。酵母杂交实验结果显示,Sc WRKY4不具有转录激活活性。实时荧光定量PCR分析表明,Sc WRKY4基因在甘蔗的根、叶、芽和皮中的表达量无明显差异,在蔗肉中的表达量最高,为对照蔗根的18.38倍;黑穗病菌侵染0~72 h,Sc WRKY4在抗病品种崖城05-179中下调表达,在感病品种ROC22中表达较稳定;受到外源激素脱落酸、水杨酸和茉莉酸甲酯以及非生物胁迫因子氯化钠和聚乙二醇胁迫后,Sc WRKY4基因均被诱导上调表达。上述研究结果表明,Sc WRKY4基因可能不参与甘蔗对黑穗病的抗性反应或在该防御方面起负调控作用,但积极响应甘蔗对盐和干旱胁迫的应答。WRKY is one of the specific transcriptional regulators in plants and widely involved in the response of plants to diverse biotic and abiotic stresses.In this study,based on our previous transcriptome data of sugarcane(Saccharum spp.),we successfully cloned a WRKY gene from sugarcane variety ROC22,named as ScWRKY4(GenBank accession number MG852087).Sequence analysis showed that the full-length cDNA of ScWRKY4 gene was 1265 bp,containing a 741 bp complete open reading frame and encoding 246 amino acids residues.ScWRKY4 protein had a conservative WRKYGQK domain and a zinc finger motif C2H2,belonging to IIc subgroup of WRKY transcription factor family.Bioinformatics analysis indicated that ScWRKY4 was an alkaline unstable hydrophilic protein with no signal peptide and transmembrane structure.The secondary structural element of ScWRKY4 protein was lack of beta helix structure.ScWRKY4 was located in the nucleus after transient expression in Nicotiana benthamiana.The result of yeast hybridization experiment demonstrated that ScWRKY4 did not possess transcriptional activity.Real-time fluorescent quantitative PCR showed that the expression of ScWRKY4 gene had no significant difference in root,leaf,bud and stem epidermis,while had the highest expression level in stem pith,which was 18.38 times higher than that in root.During inoculation with smut pathogen(Sporisorium scitamineum)at 0–72 h,the gene expression level of ScWRKY4 was down-regulated in sugarcane smut-resistant cultivar Yacheng 05-179,while was more stable in the susceptible one ROC22.The transcript of ScWRKY4 was up-regulated by exogenous hormone stresses of abscisic acid,salicylic acid,and methyl jasmonate,as well as by the abiotic treatments of sodium chloride and polyethylene glycol.These results suggest that ScWRKY4 gene may not participate in sugarcane smut resistance,or only play a negative role in this defense reaction,but positively involve in the response mechanism of salt and drought resistance in sugarcane.

关 键 词：甘蔗 WRKY转录因子 生物信息学 亚细胞定位 转录激活活性 实时荧光定量PCR 

分 类 号：S566.1[农业科学—作物学]