掌叶大黄转录因子RpMYB4基因克隆及分子表达特性

Cloning and expression characteristics of a transcription factor gene RpMYB4 in Rheum palmatum L.

MYB转录因子在植物生长发育、次生代谢及逆境胁迫等过程中发挥重要的转录调控作用。本实验在掌叶大黄转录组数据库中筛选一个包含完整开放阅读框(ORF)的MYB家族成员序列,首次克隆获得RpMYB4基因ORF,编码一条245个氨基酸的多肽,分子质量为26.99 kDa,N端含有R^(2)R3-MYB亚家族典型的2个DNA保守结合域(HTH-MYB),无信号肽或跨膜域,与其他植物物种MYB转录因子多序列比对均高达61%以上,系统进化分析显示与FtMYB8关系最近,共同聚在S4亚家族。亚细胞定位结果显示RpMYB4-GFP定位在烟草细胞核内。实时荧光定量分析表明RpMYB4的组织表达有差异,叶中表达量最高,依次为叶柄、根茎、根及种子;受200μmol·L^(-1)茉莉酸甲酯(MeJA)处理,RpMYB4表达在24 h内持续下调,200μmol·L^(-1)水杨酸(SA)处理在12和24 h显著上调,该基因对200μmol·L^(-1)脱落酸(ABA)处理未见明显变化;RpMYB4基因受干旱、高温及损伤胁迫诱导,分别在24、24、3 h处达峰值,低温胁迫抑制其表达,6 h呈谷值,其对盐胁迫响应不显著。首次获得掌叶大黄RpMYB4基因,在叶片和叶柄中表达量高,受激素SA与干旱、高温及损伤胁迫等诱导表达,为后续研究其在大黄次生代谢及逆境胁迫中的分子作用奠定基础。

MYB transcription factors play many important regulatory roles in plant growth and development,secondary metabolism,and stress adaptation processes.In this work,an MYB gene containing a complete open reading frame(ORF)was selected from the transcriptome database of R.palmatum L.RpMYB4 ORF and cloned,encoding a polypeptide of 245 amino acids with a molecular weight of 26.99 kDa.RpMYB4 lacks a signal peptide or transmembrane domain but contains two conserved DNA binding domains(HTH-MYB)of the R^(2) R3-MYB subfamily at the N-terminus.Multiple-sequence alignment demonstrated that RpMYB4 shared as high as 61%identity with many MYB proteins from other species.Phylogenetic analysis showed that RpMYB4 had the closest relationship with FtMYB8 and was clustered in the S4 subfamily.Subcellular localization by confocal microscopy showed that an RpMYB4-GFP-fusion protein localized to the nucleus in tobacco.Real-time fluorescence quantitative PCR analyses revealed that RpMYB4 was differentially expressed in various tissues,with the highest expression in leaves,followed by petioles,rhizome,and roots,and with the lowest level in mature seeds.After treatment of R.palmatum L.seedlings with 200μmol·L^(-1) MeJA,the expression of RpMYB4 in leaves was down-regulated within 24 h,and significantly up-regulated after 200μmol·L^(-1) SA treatment at 12 h and 24 h.However,gene expression did not change with 200μmol·L^(-1) ABA treatment.The transcripts of RpMYB4 under drought,high temperature,and mechanical injury stresses reached a peak at 24 h,24 h,and at 3 h,respectively,while RpMYB4 expression was inhibited by low temperature stress,reaching its lowest value at 6 h.The gene showed no significant response to salt stress.Overall,RpMYB4 was cloned from R.palmatum L.for the first time,showed high expression in leaves,and was responsive to SA and various abiotic stress treatments including drought,high temperature,and mechanical injury.The results will be useful for further analysis of secondary metabolism and stress adaptations in R.palmatum L.