文心兰15个miRNAs及其候选靶标的表达特性

Expression characteristics of 15 miRNAs and their candidate target genes in Oncidium hybridum

为了解miRNAs及其候选靶标在文心兰不同组织部位及假鳞茎受软腐病病原菌侵染中的表达规律,基于文心兰转录组和miRNA数据库进行分析,从中筛选获得15个miRNAs,结合生物信息学法对其候选靶标进行预测和功能注释,并利用实时荧光定量技术检测miRNA及候选靶标在文心兰不同组织部位和假鳞茎受软腐病病原菌侵染的表达情况.结果表明,文心兰miRNA数据库的miRNA reads分析提示15个miRNAs不同成员可能在文心兰正常植株和感病不同阶段中差异表达. psRNATarget靶标预测显示,文心兰15个miRNAs有828个候选靶标,其中67个可能与抗病相关,包括丝氨酸/苏氨酸蛋白、F-box蛋白基因、锌指蛋白、RGA抗病蛋白等.基于文心兰miRNAs读取次数(Reads)和Unigene候选靶标RPKM值,发现miRNAs及其候选靶标在文心兰正常植株和假鳞茎不同感病时段表达均存在差异.实时荧光定量PCR分析显示,在文心兰不同组织部位中,miR159a、miR167b、miR168a、miR169a、miR171a和miR172a均在假鳞茎和叶中均大量表达,并与对应候选靶标的表达呈负调控关系,推测它们参与假鳞茎和叶的发育和形态建成.在软腐病病菌侵染文心兰假鳞茎中,miR159a、miR168a、miR169a、miR171a和miR172a在8 h处理时表达量高,并且与对应的候选靶标在侵染0-8 h表达呈负调控关系,推测上述miRNAs通过负调控候选靶标在中期响应软腐病病原菌侵染过程;而miR167b在处理0h高表达,并且与候选靶标在侵染0-8h表达呈负调控关系,推测其通过下调表达参与软腐病病原菌侵染过程的响应.上述研究表明,文心兰miRNAs通过介导候选靶标的裂解可能广泛参与文心兰不同组织的发育及软腐病病原菌侵染过程的响应.

This study aimed to investigate the response mode of miRNA and candidate target genes in the disease resistance process of Oncidium. The transcriptome data and miRNA database of Oncidium leaves infected by soft rot(normal, mild,and severe infection) pathogens were analyzed. Subsequently, 15 miRNAs were screened out, and their candidate targets and functional annotations were predicted by bioinformatics analysis. A real-time quantitative PCR analysis was applied to clarify the expression of miRNAs and their candidate targets in different tissues and pseudobulbs infected by soft rot. An miRNA read analysis based on the Oncidium miRNA database suggested that different miRNAs might have different functions in normal plants and in different stages of Oncidium susceptibility. Using the psRNATarget online software to predict the candidate targets of Oncidium miRNAs, we found 828 candidate targets for 15 miRNAs in Oncidium;among them, 67 targets,including serine/threonine-protein, F-box protein gene, zinc finger protein, resistance gene analog(RGA) protein, might be related to disease resistance. Based on the miRNA reads and the reads per kilobase per million mapped reads(RPKM) values of candidate targets, the miRNAs and their candidate targets were found to be differentially expressed in normal plants and in different susceptible periods of Oncidium. A real-time quantitative PCR analysis showed that miR159 a, miR167b, miR168a,miR169a, miR171a, and miR172a were abundantly expressed in the pseudobulbs and leaves of Oncidium and were negatively correlated with the expression of the corresponding candidate targets, suggesting that they are involved in the development and morphogenesis of pseudobulbs and leaves. In the Oncidium pseudobulbs infected by soft rot pathogen, the miRNAs miR159a,miR168a, miR169a, miR171a, and miR172a were highly expressed at 8 h and showed a negatively regulated relationship with the expression of the corresponding candidate targets at 0–8 h of infection. Thus, it was speculated that the above miRNAs respond to the infection process of the soft rot pathogens in the medium term through negatively regulated candidate targets.However, miR167b was highly expressed at 0 h of infection and showed negatively regulated relationship with candidate targets at 0–8 h of infection, suggesting that it is involved in the response of soft rot pathogen infected process by down-regulating its expression. The above research shows that miRNAs might be widely involved in the development of different tissues of Oncidium by mediating the cleavage of candidate targets and response of the infection process of soft rot pathogens.