玉米NF-Y复合体的作用机制及在抗逆育种中的应用

NF-Y (nuclear factor-y) is a kind of transcription factors existing in all eukaryotic species and exhibit diversified biological functions. In plant, a number of NF-Y genes play important roles in the regulation of resistance to abiotic stresses and some processes of growth and development, and are valuable targets for crop breeding. In this project the lines of maize with transgene ZmNF-YB3、ZmNF-YA3、ZmNF-YA4 and their RNAi constructs, respectively, will be used as materials for study. The yeast-hybridization, protein coprecipitation, SDS-PAGE and tandem mass spectrometry analysis are used to identify the components of the protein complex; the Chip-sequence and whole-genome transcriptional sequence are used to look for the candidate functional elements, the key target genes, and the metabolism and/or signaling pathways that downstream genes involved in. The components of transcriptional complex of ZmNF-YB3, ZmNF-YA3 and ZmNF-YA4 involved in will be identified. The interaction and regulation of the transcriptional complex components, mechanisms of the regulation of target genes and the pathways will be discussed. New knowledge about the NF-Y complex, the differences and variety of the NF-Y family members will be accumulated in this project. The project is aimed at seeking the pivotal genes and pathways controlling the growth and development in maize to shape the strategies for the production of plants with improved tolerance to abiotic stresses and optimal yield in the fields under normal conditions.

NF-Y是真核生物中普遍存在的转录因子，参与植株对逆境胁迫的响应和生长发育的调控，是作物育种中值得重点关注的对象。本课题以分别转ZmNF-YB3、ZmNF-YA3、ZmNF-YA4基因和它们的RNAi结构的玉米株系为材料，利用酵母杂交和蛋白质免疫共沉淀、SDS-PAGE-串联质谱分析来寻找与靶基因编码蛋白互作的复合体成员，利用Chip-sequence和转录组分析来寻找靶蛋白结合的作用元件和下游基因及相关的代谢及信号转导途径。进而明确靶基因产物形成的转录调控复合体的组分，探讨个组分之间的相互作用、调控机制、作用位点、关键基因及它们调控的代谢或/和信号转导途径。通过揭示靶基因的表达改变对玉米植株抗逆性和生长发育的影响，寻找生长发育与抗逆性调控的交汇点，为玉米分子育种提供新策略和新基因。

NF-Y是真核生物中普遍存在的转录因子，参与植株对逆境胁迫的响应和生长发育的调控，是作物育种中值得重点关注的对象。本项目以获得的分别过表达或抑制表达ZmNF-YB16、ZmNF-YA1和ZmNF-YA12的转基因玉米纯合系为材料，进行基因表达、表型及生理生化性状分析及籽粒产量和抗旱性测定，寻找ZmNF-YB16基因调控的下游基因及相关的代谢及信号转导途径，初步明确了ZmNF-YB16、ZmNF-YA1在玉米抗逆机制和生长发育中的作用，确定了过表达ZmNF-YB16或ZmNF-YA1可提高玉米的抗逆性和籽粒产量，转基因株系具有很好的应用前景。同时利用酵母杂交和蛋白质免疫共沉淀、SDS-PAGE-串联质谱分析等技术寻找与靶基因编码蛋白互作的复合体成员，揭示了ZmNF-YB16和ZmNF-YC17在胞质中形成二聚体，然后进入核中与ZmNF-YA1形成三聚体，以复合体成员的形式参与转录调控，并且它们的相作受到蛋白磷酸化状态的影响。鉴定出3个与ZmNF-YB16互作的蛋白，其作用尚在鉴定。采用Chip-seq和硫尿嘧啶标记法检测新转录本等方法寻找下游基因及作用元件，得出ZmNF-YB16调节渗透胁迫下染色质核小体组装及DNA复制相关基因的初始转录和热胁迫下氧化还原基因的转录，ZmNF-YB16过表达影响了一些重要酶基因的转录及转录本丰度。在正常环境条件下，对照和转基因过表达系差异表达基因富集最显著的通路为光合作用，而在干旱胁迫下，差异表达基因显著富集的路径除光合作用外，还有与谷胱甘肽代谢和内质网蛋白质折叠相关的通路。对显著富集GO中的基因进行启动子序列分析，发现排名前四位的顺式作用元件以次为GGAGCT、CCACT、TTACA和TTATA，推测它们可能是ZmNF-YB16基因在热胁迫条件下发挥调控作用中的结合位点。在Chip-seq方法建立中，还以基因组简单的植物盐芥为材料开展了抗逆机制的研究；在玉米抗旱机制的解析中，还进行了玉米Zmbip4和ZmPTF1的基因对干旱胁迫响应的表达调控网络的研究，以资在转录组水平上相互比较，促进玉米抗旱机制与ZmNF-YB16的作用的探索。.说明：参照已发表文章，ZmNF-Y家族命名发生变化，原ZmNF-YB3改为ZmNF-YB16，ZmNF-YA4为ZmNF-YA1，ZmNF-YA3为ZmNF-YA12。