摘要
[目的]本文旨在鉴定菊花TAS3 tasi-RNA的靶基因生长素响应因子CmARF4,并探讨该基因的表达特性。[方法]以菊花品种‘神马’为材料,利用高保真PCR方法克隆CmARF4基因全长,采用5′RLM-RACE技术验证CmARF4基因是否含有TAS3剪切位点,通过qPCR分析CmARF4基因在不同组织器官中和生长素处理及盐处理条件下的表达水平。[结果]CmARF4为TAS3 tasi-RNA的靶基因,其剪切位点位于TAS35′端的第10与第11位碱基之间。CmARF4基因包含开放阅读框(ORF)2322 bp,编码773个氨基酸,预测其蛋白质相对分子质量为85.7×103,理论pI为6.44,且与刺菜蓟的同源性最高(78.63%)。在营养生长期,芽中CmARF4的表达量最高,茎中次之;在盛花期舌状花中CmARF4的表达量最高,其次是茎。亚细胞定位与转录激活活性分析结果显示:CmARF4蛋白定位在细胞核上,且无转录激活活性。CmARF4响应生长素处理,当萘乙酸(NAA)浓度为5μmol·L^-1时,CmARF4基因的相对表达量在12 h时明显升高;当NAA浓度为15μmol·L^-1时,CmARF4的相对表达量呈上升趋势。200 mmol·L^-1 NaCl处理下,CmARF4的相对表达量在盐胁迫1 h时升至最高,之后逐渐下降。[结论]CmARF4基因为TAS3 tasi-RNA的靶基因,CmARF4的转录水平受生长素及盐处理的诱导。
[Objectives]The study aimed to verify if the auxin response factor CmARF4 was a target gene of TAS3 tasi-RNA in Chrysanthemum morifolium,and to explore its expression characteristics.[Methods]The full-length of CmARF4 was obtained by high fidelity PCR using chrysanthemum variety‘Jinba’as plant material,and the cleavage site of TAS3 tasi-RNA in the CmARF4 gene was confirmed through 5′RLM-RACE PCR reactions.The relative expression levels of CmARF4 in different tissues and in response to auxin and salt treatment of chrysanthemum variety‘Jinba’were detected by qPCR.[Results]CmARF4 was a target gene of TAS3,and its cleavage site was located between the 10th and 11th bases of the 5′end of TAS3.The CmARF4 had an open reading frame(ORF)of 2322 bp encoding 773 amino acids.The predicted protein molecular weight was 85.7×103,and the theoretical isoelectric point(pI)was 6.44.Phylogenetic analysis showed that it had the closest relationship with ARF4 from Cynara cardunculus with an identity of 78.63%.CmARF4 gene had the highest expression level in bud and ray-florets during vegetative period and flowering period,followed by that in stem,respectively.Subcellular localization analysis indicated that CmARF4 protein was located in the nucleus.The CmARF4 gene had no transcriptional activity in the yeast system.The expression level of CmARF4 gene increased significantly at 12 h under 5μmol·L^-1 naphthalic acetic acid(NAA)treatment,and kept increase under 15μmol·L^-1 NAA.Under treatment with 200 mmol·L^-1 NaCl,the expression level of CmARF4 increased to the highest level at 1 h,and then gradually decreased.[Conclusions]The CmARF4 gene is the target gene of TAS3 tasi-RNA in chrysanthemum.The transcription level of CmARF4 is induced by auxin and salt treatment.
作者
时春美
朱文静
田畅
蒋甲福
宋爱萍
陈发棣
陈素梅
SHI Chunmei;ZHU Wenjing;TIAN Chang;JIANG Jiafu;SONG Aiping;CHEN Fadi;CHEN Sumei(State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Landscaping,Ministry of Agricultureand Rural Affairs/College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2020年第6期1006-1014,共9页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(31972451)
国家重点研发计划项目(2018YFD1000402)
江苏省农业科技自主创新资金项目[CX(18)2020]。
