miRNAs和mRNA基因互作网络调控小桐子抗冷性提高的分子机制

Research and development of the energy plant Jatropha curcas L. is a strategical demand for Yunnan province and our country. Low temperature is a key limiting factor for development and production of J. curcas. It is well-known that microRNAs play impotant roles in response and adaptation of plants to environmental stresses, but it is little known how these interactions between microRNAs and mRNA to integrate a network to regulate chilling resistance in plants. Based on our previous work, using two population of J. curcas with different chilling resistance as experimental material, the present proposal is going to study: 1. High-throughput sequencing, verification and comparision of transcriptome, miRomics and degradome during chill-hardening and following chilling stress in J. curcas seedlings; 2. prediction and verification of microRNA-targeted genes; 3. analysis of miRNA-mRNA interaction network during chill-hardening enhanced chilling resistance in J. curcas seedlings; and 4. cloning and functional identification of miroRNAs impotant for chilling resistance. The predicted results will explore some novel microRNAs and their target genes, as well as those micorRNAs impotant for chilling resistance, and elucidate how the miRNA-mRNA interaction gene network to regulate enhancement of chilling resistance in J. curcas and their molecular machanisms.

研究开发能源植物小桐子是云南省和国家的能源战略需求，低温是限制小桐子产量和分布的重要环境因素。已知miRNAs在植物对逆境胁迫的响应与适应中起重要作用，但miRNAs如何与mRNA互作形成网络来调控植物的抗冷性仍不清楚。基于前期工作基础，本项目拟研究：①低温锻炼及随后的低温胁迫过程中小桐子转录组、miRNA组及其降解组的高通量测序、验证与比较分析；②小桐子miRNAs靶基因的预测与验证；③低温锻炼提高小桐子抗冷性过程中miRNAs与mRNA基因互作网络的解析；④对小桐子抗冷性提高有重要作用的miRNAs的克隆与功能解析。研究结果将发掘一批新的小桐子miRNAs及其潜在的靶基因，获得对小桐子抗冷性提高有重要作用的miRNAs，并最终解析低温锻炼提高小桐子抗冷性过程中miRNAs与mRNA基因互作网络，以及这种miRNAs与mRNA的互作如何来调控小桐子抗冷性提高的分子机制。

小桐子（Jatropha curcas L.）是一种极具综合开发价值的能源植物，低温是限制小桐子产量和分布的重要环境因素。本实验室前期研究发现12°C低温锻炼能显著提高小桐子抗冷性，但这种锻炼提高小桐子抗冷性的分子机制尚未清楚。已知miRNAs在植物对逆境胁迫的响应与适应中起重要作用，但miRNAs如何与mRNA互作形成网络来调控植物的抗冷性仍不清楚。. 本研究以小桐子幼苗为材料，将26/20°C培养的植株作为对照，将一批处理植株转入12°C下进行低温锻炼48h，随后1°C低温胁迫5d;另一批未锻炼的植株直接1°C低温胁迫5d。进行小RNA组、转录组和降解组测序，以研究miRNAs和靶基因mRNA如何互作来调控小桐子的抗冷性。通过对低温锻炼及随后的低温胁迫过程中小桐子转录组、miRNA组及其降解组测序数据的进行定性和定量比对、分析和验证，获得了小桐子转录组和miRNA组变化的全景图像，鉴定到1643个已知miRNAs和330个新的miRNAs序列。低温锻炼诱导了大量差异表达miRNAs（DEMs）和差异表达基因（DEGs）参与了小桐子对低温的响应与适应过程。对小桐子miRNAs靶基因的预测与验证结果表明，这些DEMs和DEGs存在复杂的互做关系，据此构建低温锻炼提高小桐子抗冷性过程中miRNAs与mRNA的基因互作网络，在一定程度上阐明这种miRNAs与mRNA的互作如何来调控小桐子抗冷性提高的分子机制。在此基础上，挑选了对低温高响应的PP2C、CP、Cystatin、HSP70、XTH、AGO、HAT、HDAC、YABBY、ATG等基因家族进行了全基因组解析、与靶向miRNA的互作分析及其在小桐子对低温响应与适应中作用的分析，研究了小桐子低温胁迫下microRNA实时荧光定量PCR内参的筛选与比较。并扩展了在可变剪接（alternative splicing, AS）全基因组水平上如何参与低温锻炼、进而提高小桐子耐冷性形成的分子机制这一部分的内容。研究结果总体上为揭示小桐子低温锻炼下抗冷性形成的分子机制和为后续通过精准分子育种手段改良小桐子抗冷性提供了理论和实验基础。.