盐胁迫下胡杨的生理响应及miRNA表达动态变化
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摘要
胡杨(Populus euphratica Oliv.)是广泛分布于我国西北地区的重要树种,具有良好的耐旱耐盐能力,能够适应沙漠戈壁地区的恶劣环境。胡杨对于维护沙漠地区的生态平衡具有非常重要的作用,也是研究植物抗逆尤其是耐盐胁迫机理的重要模式树种。miRNA是一种短片段非编码RNA,可以与靶基因的mRNA结合后抑制或者降解目标,从而在转录后水平上起重要的调控作用。本实验以胡杨无性系为材料,研究盐胁迫条件下的生理指标变化,同时利用Hiseq高通量测序技术对不同时间点下的胡杨叶片和根部组织进行了测定,筛选出与胡杨盐胁迫相关的miRNA,分析其在不同器官和不同时间点上的表达差异。运用多元正态分布的模型对miRNA的动态表达模式进行了聚类,检验miRNA与环境的互作关系,通过靶基因的预测和功能注释,对miRNA在胡杨耐盐分子机制中发挥的作用进行了研究。取得的主要研究结果如下：
在300mmol NaCl胁迫下,胡杨叶片和根部中的Na+离子浓度,SOD酶活性,ABA含量均有不同程度的升高,在处理后6h的时候大部分生理指标变化最为显著,但K+离子浓度,MDA含量,叶绿素a、b和类胡萝卜素含量这些生理指标保持在较为稳定的水平上。结果证明胡杨对盐胁迫的生理响应是迅速有效的。
对胡杨的高通量测序结果进行分析,共发现保守miRNA290个,其中叶片中相对高表达的miRNA有75个,根部中相对高表达的有86个,其调控对象以转录因子居多。盐胁迫使得不同时间点上miRNA的表达量出现了明显的变化,显著上调的miRNA数目从11个到117个不等,显著下调的数目从23个到169个不等。对其中的高表达miRNA进行注释后结果显示,这些：miRNA的功能主要与转录因子、激素响应和ATP酶等有关。运用软件对胡杨中可能存在的新miRNA进行了预测,共获得了45个新miRNA候选序列。
运用多元正态分布模型对miRNA的动态表达数据进行聚类,结果显示根部和叶片中的miRNA在长期和短期时间段中其表达模式分别可以归纳为4-13个聚类,对这些miRNA与环境的互作关系进行了显著性检验并注释了靶基因功能,发现部分聚类中的miRNA功能与响应环境刺激和细胞死亡相关。
本研究使用了胡杨无性系作为实验材料,避免了个体基因型差异带来的影响。实验中的时间点覆盖了瞬时响应和长期适应,能反映胡杨在盐胁迫整个过程中的生理指标和miRNA表达变化。并使用了先进的统计模型来针对基因动态过程进行聚类和显著性检验,研究结果将有助于更好地理解植物复杂的耐盐分子机制。
Populus euphratica Oliv. is a unique tree species widely distributed in North-west China, for the outstanding abilities of drought and salt resistance, it can survive in the harsh environment and contributed for maintaining the ecological balance of the desert area, meanwhile the euphratica poplar is also a model tree specie for the reaseach of plant stress-resistence especially salt-resistence mechanisms. miRNA is a small non-coding RNA, regulated in post-transcription level though binding to mRNA to inhibit or degrade the target. In this experiment we used clones of P. euphratica as material to study the change of physiological indexs, then sequenced the leaves and roots samples at different time points to find salt-response miRNA, to investigate the expression differences between organs and time. We used multivariate normal distribution based model to cluster dynamic expressed miRNA, then predicted and annotated its'target gene. The role of miRNA in salt stress responsed P. euphratica was discussed. We obtained the following results:
(1) Na+concentrations, SOD activities and ABA contents rised after300mmol NaCl treatment, the physiological indexs changed significantly at6h, but K+concentrations, MDA, chlorophyll a, b and carotenoid contents remained steadily. The result indicated P. euphratica had a quickly and efficiently physiological response for salt stress.
(2) After analized sequencing result, we found290conserved miRNA,75of which were leaf-specific high expressed,86were root-specific high expressed, the mainly regulated targets were transcription factors. miRNA expressions have been significantly changed at different time points as salt stressed, numbers of up-regulated miRNA varied from11to117, numbers of down-regulated miRNA varied from23to169. After annotated some high expressed miRNA, we found the miRNAs'target gene were transcription factors, hormone response factors and ATPases et al. Then we predicted some novel-miRNAs by software, obtained45novel miRNA candidates.
(3) We used multivariate normal distribution based model to cluster dynamic expressed miRNA, the result showed cluster number of miRNA counts from4to13clusters, then we tested the significance of gene-enviroment interaction and annotated the miRNA, the miRNA annotations of some clusters were mainly related to response to environmental stimulus and cell death.
For the first time we used clones of P. euphratica as matearials for miRNA reserch, which can avoid interferences come from samples'different genetic background. Timescale of experiment including short-term shock phase and long-term adaption phase, which can reflect the physiological and miRNA expression changes in entire time couse. We then used advanced statistic model to cluster genes. This result will benifit further reserch for plant salt-resistence mechanisms.
在300mmol NaCl胁迫下,胡杨叶片和根部中的Na+离子浓度,SOD酶活性,ABA含量均有不同程度的升高,在处理后6h的时候大部分生理指标变化最为显著,但K+离子浓度,MDA含量,叶绿素a、b和类胡萝卜素含量这些生理指标保持在较为稳定的水平上。结果证明胡杨对盐胁迫的生理响应是迅速有效的。
对胡杨的高通量测序结果进行分析,共发现保守miRNA290个,其中叶片中相对高表达的miRNA有75个,根部中相对高表达的有86个,其调控对象以转录因子居多。盐胁迫使得不同时间点上miRNA的表达量出现了明显的变化,显著上调的miRNA数目从11个到117个不等,显著下调的数目从23个到169个不等。对其中的高表达miRNA进行注释后结果显示,这些：miRNA的功能主要与转录因子、激素响应和ATP酶等有关。运用软件对胡杨中可能存在的新miRNA进行了预测,共获得了45个新miRNA候选序列。
运用多元正态分布模型对miRNA的动态表达数据进行聚类,结果显示根部和叶片中的miRNA在长期和短期时间段中其表达模式分别可以归纳为4-13个聚类,对这些miRNA与环境的互作关系进行了显著性检验并注释了靶基因功能,发现部分聚类中的miRNA功能与响应环境刺激和细胞死亡相关。
本研究使用了胡杨无性系作为实验材料,避免了个体基因型差异带来的影响。实验中的时间点覆盖了瞬时响应和长期适应,能反映胡杨在盐胁迫整个过程中的生理指标和miRNA表达变化。并使用了先进的统计模型来针对基因动态过程进行聚类和显著性检验,研究结果将有助于更好地理解植物复杂的耐盐分子机制。
Populus euphratica Oliv. is a unique tree species widely distributed in North-west China, for the outstanding abilities of drought and salt resistance, it can survive in the harsh environment and contributed for maintaining the ecological balance of the desert area, meanwhile the euphratica poplar is also a model tree specie for the reaseach of plant stress-resistence especially salt-resistence mechanisms. miRNA is a small non-coding RNA, regulated in post-transcription level though binding to mRNA to inhibit or degrade the target. In this experiment we used clones of P. euphratica as material to study the change of physiological indexs, then sequenced the leaves and roots samples at different time points to find salt-response miRNA, to investigate the expression differences between organs and time. We used multivariate normal distribution based model to cluster dynamic expressed miRNA, then predicted and annotated its'target gene. The role of miRNA in salt stress responsed P. euphratica was discussed. We obtained the following results:
(1) Na+concentrations, SOD activities and ABA contents rised after300mmol NaCl treatment, the physiological indexs changed significantly at6h, but K+concentrations, MDA, chlorophyll a, b and carotenoid contents remained steadily. The result indicated P. euphratica had a quickly and efficiently physiological response for salt stress.
(2) After analized sequencing result, we found290conserved miRNA,75of which were leaf-specific high expressed,86were root-specific high expressed, the mainly regulated targets were transcription factors. miRNA expressions have been significantly changed at different time points as salt stressed, numbers of up-regulated miRNA varied from11to117, numbers of down-regulated miRNA varied from23to169. After annotated some high expressed miRNA, we found the miRNAs'target gene were transcription factors, hormone response factors and ATPases et al. Then we predicted some novel-miRNAs by software, obtained45novel miRNA candidates.
(3) We used multivariate normal distribution based model to cluster dynamic expressed miRNA, the result showed cluster number of miRNA counts from4to13clusters, then we tested the significance of gene-enviroment interaction and annotated the miRNA, the miRNA annotations of some clusters were mainly related to response to environmental stimulus and cell death.
For the first time we used clones of P. euphratica as matearials for miRNA reserch, which can avoid interferences come from samples'different genetic background. Timescale of experiment including short-term shock phase and long-term adaption phase, which can reflect the physiological and miRNA expression changes in entire time couse. We then used advanced statistic model to cluster genes. This result will benifit further reserch for plant salt-resistence mechanisms.
引文
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