中国农业科学 ›› 2018, Vol. 51 ›› Issue (23): 4409-4423.doi: 10.3864/j.issn.0578-1752.2018.23.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

一个甘蔗Ⅱd类WRKY转录因子基因的克隆和表达分析

张旭(),凌辉,刘峰,黄宁,王玲,毛花英,李聪娜,汤翰臣,苏炜华,苏亚春,阙友雄()   

  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/福建农林大学教育部作物遗传育种与综合利用重点实验室,福州 350002
  • 收稿日期:2018-06-12 接受日期:2018-07-29 出版日期:2018-12-01 发布日期:2018-12-12
  • 基金资助:
    国家自然科学基金(31671752);国家自然科学基金(31101196);福建省杰出青年基金(2015J06006);福建省高校杰出青年科研人才计划项目苏亚春-2017;国家农业产业技术体系项目(CARS-17)

Cloning and Expression Analysis of a Ⅱd Sub-Group WRKY Transcription Factor Gene from Sugarcane

ZHANG Xu(),LING Hui,LIU Feng,HUANG Ning,WANG Ling,MAO HuaYing,LI CongNa,TANG HanChen,SU WeiHua,SU YaChun,QUE YouXiong()   

  1. Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture, Fujian Agriculture and Forestry University/Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2018-06-12 Accepted:2018-07-29 Online:2018-12-01 Published:2018-12-12

摘要:

【目的】WRKY是植物特有的一类转录因子,在生理调控和逆境响应过程中有着重要作用。通过分析WRKY转录因子基因在甘蔗生长发育及抗逆境过程中的作用,为甘蔗抗逆分子育种提供优良的基因资源。【方法】从甘蔗转录组数据库中挖掘到一条WRKY基因Unigene序列,并通过RT-PCR扩增得到cDNA全长序列,利用ORF finder、Smart、ExPaSy、Prabi、NetPhos、Cell-PLOC 2.0和DNAMAN6.0软件对该基因序列及其编码蛋白序列进行生物信息学分析,并使用MEGA6.0软件构建系统进化树;构建pCAMBIA1300-WRKY-GFP融合表达载体,通过农杆菌转化本氏烟(Nicotiana benthamiana),确定WRKY蛋白在烟草叶片中的亚细胞定位;利用酵母杂交试验验证WRKY是否具有转录自激活活性;采用实时荧光定量PCR(qRT-PCR)方法分析WRKY在甘蔗品种ROC22中的组织特异性(根、蔗芽、叶、蔗髓和皮)及其在MeJA(100 μmol·L -1)、SA(5 mmol·L -1)、PEG(25%)、NaCl(250 mmol·L -1)、CuCl2(500 mmol·L -1)和CdCl2(500 mmol·L -1)胁迫条件下表达量的变化。【结果】从甘蔗品种ROC22中克隆获得一个WRKY转录因子基因,命名为ScWRKY6(GenBank登录号为MH393927)。该基因序列全长1 289 bp,包含1个1 059 bp的ORF,编码352个氨基酸,并具有45个磷酸化位点;理论等电点为9.73,不稳定指数为50.23,亲水性为-0.579,推测其为碱性不稳定亲水性蛋白。ScWRKY6蛋白具有1个WRKY结构域和1个锌指结构域(CX5CX23HXH),该蛋白氨基酸序列与高粱(Sorghum bicolor)WRKY(XP_002464211.1)的同源性最高,根据系统进化树分析可以推测其属于WRKY家族Ⅱd亚类。亚细胞定位结果显示,ScWRKY6::GFP融合蛋白定位在细胞核上。酵母转录激活验证试验显示,ScWRKY6蛋白不具有转录自激活活性。qRT-PCR分析表明,ScWRKY6在甘蔗中为组成型表达,其表达量由大到小依次为蔗芽、叶、根、皮和蔗髓,其中蔗芽、叶和根的表达量分别为蔗髓的2.05、1.55和1.37倍。ScWRKY6在NaCl、PEG、MeJA、重金属Cu 2+和Cd 2+胁迫诱导下表达量均上调,其在NaCl处理12 h时表达量最高,为对照的4.18倍;在PEG处理3 h时表达量最高,为对照组的6.88倍;在CuCl2和CdCl2诱导24 h时表达量最高,分别为对照组的3.63倍和4.86倍。【结论】ScWRKY6蛋白定位在细胞核上,不具有转录自激活活性;该基因在甘蔗不同组织部位中均有表达,且受NaCl、PEG、CuCl2和CdCl2等胁迫的诱导,推测ScWRKY6可能在甘蔗干旱应答、耐盐及金属离子胁迫响应中发挥作用。

关键词: 甘蔗, WRKY转录因子, 外源胁迫, 实时荧光定量PCR

Abstract:

【Objective】 WRKY, a group of unique transcription factors in plants, plays an important role in plant physiological regulation and stress response. Through analysis of the role of transcription factor WRKY in sugarcane growth and development and stress resistance, this study will provide excellent gene resources for sugarcane resistance molecular breeding. 【Method】 A unigene sequence of WRKY gene was extracted from the sugarcane transcriptome database, and its full-length cDNA sequence was obtained by RT-PCR amplification. Bioinformatics analysis of this gene sequence and its encoded protein sequence was performed using ORF finder, Smart, ExPaSy, Prabi, NetPhos, Cell-PLOC 2.0 and DNAMAN6.0 softwares, and the phylogenetic tree analysis was constructed using MEGA6.0 software. The fusion expression vector of pCAMBIA1300-WRKY-GFP was constructed and delivered into Nicotiana benthamiana by Agrobacterium mediated method to determine the subcellular localization of WRKY protein in tobacco leaves. Yeast hybridization assay was used to verify whether WRKY possess transcriptional self-activation activity. The tissue specific expression (root, bud, leaf, stem pith and epidermis) of WRKY and its dynamic expression under MeJA (100 μmol·L -1), SA (5 mmol·L -1), PEG (25%), NaCl (250 mmol·L -1), CuCl2 (500 mmol·L -1) and CdCl2 (500 mmol·L -1) stresses in sugarcane variety ROC22 were analyzed by quantitative real-time PCR (qRT-PCR) technique.【Result】 A WRKY transcription factor gene, named ScWRKY6 (GenBank Accession Number: MH393927), was cloned from the sugarcane variety ROC22. This gene sequence was 1 289 bp in full length with a 1 059 bp ORF, encoding 352 amino acids, and contained 45 phosphorylation sites. The theoretical isoelectric point, the instability index and the hydrophilicity of ScWRKY6 protein was 9.73, 50.23 and -0.579, respectively, which is supposed to be an alkaline unstable hydrophilic protein. The ScWRKY6 protein has one WRKY domain and one zinc finger motif (CX5CX23HXH), and its amino acid sequence has the highest homology with Sorghum bicolor WRKY (XP_002464211.1). It is speculated that this gene belongs to the Ⅱd sub-group of WRKY family according to phylogenetic tree analysis. Subcellular localization results showed that the ScWRKY6::GFP fusion protein was located in the nucleus. Yeast transcriptional activation verification experiments indicated that ScWRKY6 protein did not have transcriptional auto-activation activity. qRT-PCR analysis revealed that ScWRKY6 was constitutively expressed in sugarcane, and the expression level in order from high to low were in bud, leaf, root, stem epidermis and stem pith. Its expression in bud, leaf and root were 2.05, 1.55 and 1.37 times higher than that in stem pith, respectively. The expression level of ScWRKY6 was up-regulated under the stresses of NaCl, PEG, MeJA, Cu 2+ and Cd 2+. Its highest expression was 4.18, 6.88, 3.63, 4.86 times higher than of the control when treated with NaCl for 12 h, PEG for 3 h, CuCl2 for 24 h and CdCl2 for 24 h, respectively.【Conclusion】 ScWRKY6 protein was located in the nucleus and did not have transcriptional auto-activation activity. The gene was expressed in different sugarcane tissues and was induced by NaCl, PEG, CuCl2 and CdCl2 treatments. It is presumed that the ScWRKY6 may play a role in response to drought stress, salt tolerance and metal ion stress in sugarcane.

Key words: sugarcane, WRKY transcription factor, exogenous stress, qRT-PCR