茶树miR398家族的鉴定及在非生物胁迫和激素处理下的表达

Genome-wide identification and expression analysis of miR398 family under abiotic stress and phytohormone treatment in tea plant

MicroRNA398(miR398)是植物中一类高度保守的miRNA成员,为了解茶树miR398(csn-miR398)家族成员结构特点、进化特性及在非生物胁迫和水杨酸(salicylic acid,SA)处理下的调控作用,对茶树miR398序列进行多重比对和进化特性分析、靶基因预测、表达模式分析及RNA寡核苷酸(RNA oligonucleotides)过表达验证.结果显示,茶树含有8个miR398家族成员,系统进化树分析显示茶树miR398家族成员均位于其前体3ʹ端臂,并且茶树miR398f/b-1保守性低于茶树miR398-/a/a-3p/b-2/b-3/b-3p.序列多重比对显示茶树miR398序列长度均为21 nt,并且与其他植物相比,茶树miR398序列第2位和第19位核苷酸发生更多突变事件.靶基因预测结果表明,茶树miR398家族成员可能靶向CsCSD1、CsCSD2和CsCCS.过表达csn-miR398a-3p负调控CsCSD1表达.启动子分析显示CsMIR398基因含有光响应、胁迫响应和SA响应元件.荧光定量PCR结果显示多数茶树miR398表达量在干旱胁迫呈下降趋势,低温胁迫呈上升趋势,SA处理下csn-miR398-/a-3p/b-2/b-3p在12 h达到最大值.此外,csn-miR398a-3p仅与CsCSD1在干旱和低温处理下呈相反表达模式.本研究表明miR398家族成员成熟体序列高度保守,并且csn-miR398a-3p和CsCSD1可能在茶树应对干旱和低温胁迫具有重要意义.(图5表2参47)

MicroRNA398(miR398)belongs to a highly conserved miRNA family.However,no study has focused on the identification and analysis of miR398 in tea plants(csn-miR398).The purpose of this study was to understand the structural features and evolutionary characteristics of members of the tea plant miR398 family and their regulatory effects under abiotic stress and salicylic acid(SA)treatment.We analyzed the sequences of plant miR398 and phylogenetically analyzed tea plants and other plants.We also predicted the target of tea plant miR398 and displayed the expression patterns of tea plant miR398 and their target genes,as well as RNA oligonucleotides for expression verification.Eight csn-miR398s were identified.A phylogenetic tree revealed that they were all located at the 3′end of their precursor,and csn-miR398f/b-1 maintained lower conservation than csn-miR398-/a/a-3p/b-2/b-3/b-3p in the evolutionary process.Multiple sequence alignments revealed that the length of the tea plant miR398 was 21 nucleotides.In addition,the variation intensity of the second and nineteenth nucleotides for tea plant miR398 sequences was higher than that for other plants.Target gene prediction analysis revealed csn-miR398s targeted CsCSD1,CsCSD2,and CsCCS.Overexpression of csnmiR398a-3p negatively regulated CsCSD1 expression.Furthermore,numerous light-responsive,stressresponsive,and SA responsive-related cis-acting elements were evident in CsMIR398.Quantitative real-time polymerase chain reaction revealed that most csn-miR398s were reduced under drought stress but induced under cold stress,and csn-miR398-/a-3p/b-2/b-3p reached a peak after 12 h of SA treatment.We found that csn-miR398a-3p negatively regulated the expression of CsCSD1 under cold and drought stresses.The findings indicate that the tea plant miR398 family is highly conserved and that csn-miR398a-3p and CsCSD1 are important in responses of tea plants to drought and cold stresses.