microRNA调控铝诱导花生根尖细胞程序性死亡发生的分子机制

Programmed cell death(PCD) and microRNAs(miRNAs) play important roles in growth, development and stress adaption in crops. Our previous results showed that PCD could be induced by aluminum(Al), and there was a negative relationship between PCD production and Al tolerance. However, it is not known about the effect and mechanism of miRNA regulating Al-induced PCD. In the present study, peanut cultivars Zhonghua 2 (Al sensitive) and 99-1507(Al-resistant) are used for investigating. Firstly, total RNA from the control and Al treatment will be isolated to construct libraries for miRNA sequencing, and construct miRNA differential expression profiles. Secondly, we will use Degradome sequencing method to reveal miRNA target genes. The differentially expressed genes will be analyzed by GO annotation and KEGG pathway analysis. Thirdly, 1-2 candidate genes of the pre-miRNA and its target gene will be cloned, the vectors of interference and overexpression are constructed, and these miRNAs and its target genes will be silenced or over-expressed in Arabidopsis or peanut to evaluate their function by Agrobacterium mediated transformation. The expressions of miRNAs and its target gens are analyzed by RT-PCD and Northern blot. In the meanwhile, the changes of Al-induced PCD in transgenic plants will be analyzed. The results, combined our present researches, it should reveal the effects and regulating mechanism of miRNA on Al-induced PCD by regulating its target genes such as SOD to control ROS signal or regulating its target gene expression directly such as caspase-like and AhSAG .

细胞程序性死亡(PCD)和miRNA在作物生长发育、环境胁迫应答中发挥重要作用，我们首先发现铝诱导花生根尖PCD及其与耐铝能力负相关现象，但对miRNA在铝诱导PCD发生中的调控作用及机制尚未见报告。本项目拟以铝敏感花生品种中花2号和耐铝品种99-1507为材料，进行以下研究：首先,分离对照和铝处理根尖总RNA，进行miRNA测序，构建miRNA差异表达谱。其次，利用降解组测序筛选miRNA靶基因，进行GO功能注释，KEGG代谢通路分析。第三，克隆1-2个候选miRNA前体及其靶基因，构建干扰及过表达载体，利用农杆菌介导法导入拟南芥或花生中，利用RT-PCR和Northern blot等分析转基因植株miRNA及靶基因表达，以及铝诱导PCD发生水平变化。阐明miRNA可能通过调节超氧化物歧化酶等，控制ROS信号，或直接作用于类Caspase、AhSAG等，从而调节铝诱导PCD发生的机制。

细胞程序性死亡（PCD）和miRNA在作物生长发育、环境胁迫应答中发挥重要作用，通过研究miRNA在铝诱导花生根尖细胞程序性死亡发生中的分子机制，获得主要结果如下：.1．鉴定了花生miRNA，发现大量新的和未知miRNA。本研究鉴定出2284个miRNA，分成4类：已知miRNA、新miRNA、潜在的新miRNA、未知miRNA，较前人鉴定的1082个，增加了一倍多，这些miRNA分属49个家族，长度为24 nt的最多。.2．找到铝胁迫响应相关miRNA。在耐铝品种（99-1507）、敏感品种（ZH2）中分别鉴定到45、67个铝胁迫响应相关的miRNA，其中35个为二者所共有，miRNA上调表达趋势明显。.3.预测获知miRNA靶基因。鉴定到749个miRNA靶向19237个靶基因，包括147个差异表达miRNA靶向的8192个靶基因，其中，6724个基因被注释到4387个GO术语；筛选出89对miRNA-mRNA，推测它们可能通过相互作用，参与铝诱导PCD发生调控。.4．验证了miR482a、miR398等部分重要miRNA的功能。过表达花生miR482a-3p、miR398的拟南芥种子的长、宽及百粒重均增加；在低浓度（50 μmol/L）铝胁迫下，过表达miR482a-3p拟南芥的相对根伸长率高于对照，耐铝性增强；miR398正义转基因拟南芥的相对根伸长率显著高于反义及野生型，缓解铝毒害对拟南芥根伸长生长的抑制作用。.5．AhERF13为miro2916的下游靶标，具有自激活活性。AhMUG可通过互作，正调靶基因AhMC1。AhUB4抑制茎增粗和叶片生长，降低MC1表达。.6. 建立花生花萼管通道遗传转化新技术，阳性率达到50%，明显高于前人报道结果。.7.AhBI-1基因促进花生根系和地上部分的生长，过表达AhBI-1基因种子萌发率、幼苗生长速度、根系生长显著优于野生型。