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
出 处:《中国农业科学》2018年第23期4409-4423,共15页Scientia Agricultura Sinica
基 金:国家自然科学基金(31671752和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))
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(100μmol·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 059bp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、Me【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, Ex Pa Sy, Prabi, Net Phos, 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(Gen Bank 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(CX5 CX23 HXH), and its amino acid sequence ha
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