| 摘要: |
| 【目的】探究文心兰miR168的分子进化特性，揭示文心兰miR168a及其候选靶基因响应软腐病菌侵染的表达模式。【方法】以文心兰miRNA文库、转录组数据库及miRbase数据库获取的植物miR168前体和成熟体序列为研究对象，利用生物信息学方法对其进行系统进化树构建、保守性分析、前体二级结构分析及靶基因预测；通过PCR技术克隆文心兰miR168a前体，采用qRT-PCR技术分析miR168a及其候选靶基因在文心兰不同组织（根、叶、假鳞茎）和感染软腐病（0，12，24，36和48 h）不同时间的表达模式。【结果】植物miR168序列保守性较高，来源于miR168前体的5p臂保守性高于3p臂，miR168前体分歧较大，茎序列的保守性高于环序列；从文心兰中克隆得到93 bp miR168a前体序列，包含21 nt miR168a成熟体序列。进化分析表明，物种亲缘关系对其前体和成熟体序列的形成影响较小；二级结构显示，文心兰miR168a前体能形成典型的发卡结构，其成熟体位于前体的5p臂；随着软腐病侵染时间延长，文心兰miR168a与其候选靶标RNA剪切复合体核心蛋白（AGO1）、RPM1互作蛋白（RIN4）、羧酸酯酶（CXE8）的表达趋势基本呈负相关，而与其候选靶标RNA解旋酶（PRH47A）、泛素羧基末端水解酶（UCH）的表达趋势呈正相关，说明可能存在其他miR168家族成员或miRNAs同时调控。【结论】文心兰miR168与其他植物miR168进化上兼具保守性和特异性，miR168a上调表达可能会增强文心兰的抗病性。 |
| 关键词: 文心兰 miR168 进化特征 软腐病 候选靶标基因 |
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| 基金项目:福建省重大农业科技专项（2015NZ0002-1）；福建农林大学科技创新基金项目（CXZX2019037S，KF2015108） |
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| Molecular evolutionary characteristics of miR168 and its response patterns to soft rot infection in Oncidium hybridum |
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LIN Zhengchun,LAI Zhongxiong,WU Qiuzhen,et al
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| Abstract: |
| 【Objective】This study explored the evolutionary characteristics of miR168 family in Oncidium hybridum response and analyzed the expression patterns of oh-miR168a and its candidate target genes to soft rot infection.【Method】Plant miR168 mature (miRNA) and precursor (pre-miRNA) sequences obtained from miRbase database,Oncidium hybridum miRNA library and transcriptome were used to conduct phylogenetic tree,analyze secondary structure and predict target genes by bioinformatics analysis.Pre-miR168a sequence of Oncidium hybridum was cloned by PCR technology.Quantitative real-time PCR (qRT-PCR) was employed to analyze the expression levels of miR168a in different tissues including roots,leaves and pseudobulbs.The expression levels of miR168a and its possible targets of Oncidium hybridum 0,12,24,36 and 48 h post inoculation were also investigated.【Result】Mature miR168 sequences were moderately conserved in plant species,miR168 derived from the 5-terminal arm of pre-miR168 was highly conserved,whereas the 3-terminal arm was quite specific.Precursor miR168 sequences were diversified and the stem sequences of pre-miR168 were more conserved than those of loops.Pre-miR168a sequence of Oncidium hybridum was 93 bp with 21 nt mature sequence.The phylogenetic relationship showed that species specificity was not a major cause of the clustering results for oh-miR168a.Pre-miR168a spontaneously formed a typical,stable hairpin secondary structure,and the mature miR168a sequence was located in the 5-terminal arm.The miR168a displayed negative correlation with expressions of target genes of AGO1,RIN4 and CXE8,while it had no negative correlation with that of PRH47A and UCH,suggesting possible simultaneous regulation be other miR168 family members or miRNAs.【Conclusion】The miR168 family members were conserved and species-specific, and up-regulation of miR168a might enhance resistance to soft rot infection in Oncidium hybridum. |
| Key words: Oncidium hybridum miR168 evolutionary characteristics soft rot predict target gene |
