西瓜CGMMV胁迫下MIR319家族成员及靶基因的鉴定分析

Identification of MIR319 Family Members and Their Target Genes in Response to Cucumber Green Mottle Mosaic Virus Infection in Watermelon

为明确MIR319家族成员(miR319、miR319a及miR319a-3p)在黄瓜绿斑驳花叶病毒(CGMMV)胁迫应答中的作用,本研究将MIR319的成熟序列与西瓜基因组进行Blast比对获得其前体基因,利用MEGA对前体基因进行系统进化分析,PlantCARE对前体基因启动子区的顺式作用元件进行分析,降解组测序对MIR319的靶基因进行分析,转录组测序及qRT-PCR分析MIR319靶基因的表达模式。研究结果获得MIR319家族成员共同前体基因Pre-MIR319,其长度为170 bp,含有完整的茎环结构;序列比对显示,MIR319家族成熟序列在5′端第2~第14位碱基高度保守;西瓜Pre-MIR319与35个物种的116条miR319前体序列经系统进化分析后分为四个分支,其中西瓜Pre-MIR319与马铃薯miR319a前体基因(MI0025952)距离最近;Pre-MIR319启动子区含有光响应元件、赤霉素响应元件、乙烯响应元件、茉莉酸甲酯响应元件、MYB和MYC等多个顺式作用元件;降解组测序获得MIR319家族成员的5个靶基因,其中Cla019567、Cla013523、Cla023342和Cla002428注释为TCP转录因子,Cla013668注释为MYB转录因子,剪切位点位于MIR319家族成员成熟序列5′端的第10位碱基;靶基因编码氨基酸数目为319~554 aa、分子量为34.94~61.21 kDa、理论等电点为5.29~7.80,定位于细胞核/细胞质中,均不含跨膜结构域;转录组测序及qRT-PCR分析表明miR319a负调控靶基因Cla013523(TCP)的表达。本研究结果进一步明确了MIR319家族成员在CGMMV胁迫应答中的作用及其对靶基因的调控作用。

In order to reveal the possible roles of MIR319 family(miR319,miR319 a and miR319 a-3 p)under the CGMMV stress,mature sequences of MIR319 were blasted against the watermelon genome to obtain the precursor gene.MEGA was used to analyze the evolutional relationship of precursor genes for MIR319.PlantCARE was used to analyze the cis-acting regulatory elements of the precursor gene promoter.Degradome sequencing was used to identify target genes of MIR319,and transcriptome sequencing and qRT-PCR were used to obtain the expression of target genes.The common precursor gene of MIR319,Pre-MIR319 was obtained,which was 170 bp in length,able to form the stem-loop structure.Sequence alignment showed that mature sequences of MIR319 were highly conserved at the 2~14 bases of 5′-terminal.Phylogenetic analysis of watermelon Pre-MIR319 and 116 miR319 precursor sequences from 35 species divided these precursor sequences into four branches.Watermelon Pre-MIR319 was closest to potato precursor gene miR319a(MI0025952).The promoter of Pre-MIR319 contain several cis-acting regulatory elements,such as light responsive element,gibberellin responsive element,ethylene responsive element,methyl jasmonate response element,MYB,MYC and etc.Five target genes of MIR319 family,Cla019567,Cla013523,Cla023342,Cla002428 and Cla013668,were predicted by degradome sequencing results.Among which,Cla019567,Cla013523,Cla023342 and Cla002428 are annotated as TCP transcription factor and Cla013668 is annotated as MYB transcription factor.The cleavage sites were located at the 10th of MIR319 at the 5′-terminal end.The amino acids number,molecular weight and theoretical isoelectric point of target genes were 319~554 aa,34.94~61.21 kDa and 5.29~7.80,respectively.These proteins do not contain transmembrane domains and was located in the nucleus/cytoplasm.Expression profiles of target genes using transcriptome and qRT-PCR analysis revealed that miR319 a negatively regulated its target gene Cla013523(TCP).These results clarified the role of MIR319 family members in CGMMV stress response and revealed the regulation of MIR319 on their target genes.