玉米幼胚胚性愈伤组织诱导过程中重要miRNA及靶转录因子的功能研究

Introduction of embryonic callus is an essential process in maize transgenic breeding. Low introduction efficiency for most of maize elite inbred lines prevents maize transgenic breeding from rapid development. In our previous study, the high-introduction effeiciency inbred line 18-599R was conducted to small RNA deep sequencing. As a result, 4 miRNA families were displayed to sharply down-regulated during the embryonic callus introduntion. Three transcript factors were respectively decided to be the candidate target genes of the four miRNAs by bioinformatic prediction and digital gene expression profiling. Further analysis revealed that the loci of the transcript factor genes is consistent with the QTL controlling the introduction of maize embryonic callus identified in the previous research. In the project, the accurate cutting site of target mRNA by the miRNAs will be identified using 5'RACE. The function of the miRNAs and targets involved in embryonic callus induction will be determined by embryo-spesific overexpression of the miRNAs and RNAi of the transcript factor genes. In addition, the interaction genes of the three transcript factors will be genome-widely revealed by ChIP-Seq technique. Then the regulatory network involved in the process of maize embryonic callus introduction will be constructed according to gene annotations.

幼胚胚性愈伤组织诱导是玉米转基因育种的重要技术环节，而我国具有高胚性愈伤诱导率的骨干自交系极少，阻碍了转基因玉米育种的快速发展。前期研究以高胚性愈伤诱导率自交系18-599R为材料，通过深度测序挖掘了4个在胚性愈伤诱导整个过程中急剧下调的miRNA家族，经靶基因预测结合数字表达谱分析和qRT-PCR验证结果，确定了3个转录因子作为以上4个miRNA家族的候选靶基因，而这3个转录因子基因在染色体上的位置与前期定位的胚性愈伤诱导率QTL位点是一致的。本项目拟首先采取5'RACE技术对miRNA剪切靶转录因子的精确位点进行鉴定；同时分别利用胚特异过表达和RNAi技术对以上miRNA和转录因子进行功能验证，获得控制玉米胚性愈伤诱导过程的miRNA和转录因子基因；并通过ChIP-Seq在全基因组水平上挖掘靶转录因子的互作基因，结合基因功能注释构建由miRNA介导的幼胚胚性愈伤诱导过程的分子调控网络。

幼胚胚性愈伤组织诱导是玉米转基因育种的重要技术环节，而具有高胚性愈伤诱导率的骨干自交系极少，阻碍了转基因玉米育种的快速发展。前期研究以高胚性愈伤诱导率自交系18-599R 为材料，通过miRNA深度测序和降解组测序挖掘了4 个在胚性愈伤诱导整个过程中急剧下调的 miRNA 家族和3个靶向的转录因子。本项目通过5’RACE技术成功鉴定了miR159a和miR167的靶基因ZmMYB138和ZmARF34的精确剪切位点；通过转基因过表达验证了miR159a和miR166的下调表达在玉米幼胚胚性愈伤组织诱导过程中的调控作用；进而利用突变体和转基因方法验证了靶基因ZmMYB138和ZmARF34对玉米幼胚脱分化的正向贡献；通过生物学实验验证了胚性愈伤组织诱导过程中ZmMYB138通过抑制GA信号传导过程来阻止幼胚分化产生不定芽，从而进一步促进脱分化的作用；建立了由miR159a和miR167介导的玉米幼胚脱分化过程的调控途径。为培育高胚性愈伤诱导率骨干系，加快转基因玉米育种进程奠定基础。本项目发表学术论文4篇，其中3篇SCI收录论文累计影响因子17.5，单篇最高影响因子11.95；授权发明专利1项、实用新型专利4项；培养四川省学术带头人后备人选1名、博士研究生2名、硕士研究生4名。