玉米ZmDi19-7基因响应干旱胁迫的分子机制研究

Drought is the most devastative abiotic stress that severely affects the growth and development of maize. In our previous research, one gene, named ZmDi19-7, was cloned from drought-tolerant maize inbred lines. This gene was strongly up-regulated when maize was under drought stress. Additionally, transgenic Arabidopsis thaliana (Col ecotype), carrying an over-expression plasmid with a full length cDNA of ZmDi19-7, exhibited enhanced drought tolerance. However, the biological function, together with the molecular mechanism for the anticipating role in drought tolerance for ZmDi19-7 are still not clear. In current proposal, we want to acquire two kinds of transgenic maize plants, one can over-express the target genes, the other carries an CRISPR/Cas9 plasmid, which could knock out target gene specifically. Afterwards, we could detailedly quantify the phenotype of both transgenic maize plants, as well as determine various physiological indexes that indicates the ability of drought tolerance. This would help us to clarify the ability for drought tolerance of the target gene in maize. Moreover, the difference of transcriptome between drought-tolerant transgenic and control maize plants will be determined with high-throughput sequencing technology, which would facilitate to identify potential key regulatory genes. Furthermore, yeast two-hybrid, together with co-immunoprecipitation technology will be applied to identify the protein that can directly interact with ZmDi19-7. Taken together, the strategy in present research will help us to acquire a modest comprehensive picture of the molecular mechanism for the drought tolerance function for ZmDi19-7 in maize. The present research will have a great practical effect on creating drought-tolerant maize breeds for breeding programs in future.

干旱是影响玉米生长发育最主要的逆境因子之一。本项目组从玉米中克隆了一个响应干旱胁迫的基因ZmDi19-7。在受到干旱胁迫的玉米中，该基因显著上调表达；并且，过量表达该基因的转基因拟南芥抗旱性也显著增强。但该基因在玉米中的抗旱生物学功能以及响应干旱胁迫的分子机制还不清楚。本研究拟通过创制ZmDi19-7基因的过量表达和敲除的转基因玉米株系，分析转基因玉米株系的抗旱表型、测定抗旱相关的生理生化指标，以明确玉米ZmDi19-7基因响应干旱胁迫的生物学功能。同时，利用转录组测序技术，分析转基因植株与非转基因植株的差异表达基因，解析ZmDi19-7基因响应干旱胁迫的信号通路；结合酵母双杂交、免疫共沉淀等技术筛选、鉴定ZmDi19-7的互作蛋白，解析玉米ZmDi19-7基因响应干旱胁迫的分子机制。本项目的开展丰富了抗旱分子机制的研究，对于培育抗旱玉米新品种具有重要的指导意义和实际应用价值。

玉米是我国第一大粮食和饲料作物，其生长发育和产量受逆境胁迫影响较大，频繁发生的逆境灾害给我国的玉米生产造成了巨大损失，挖掘玉米基因组中响应多种胁迫的基因及培育抗逆新品种是应对逆境胁迫最为经济有效的方式。本项目组前期从玉米中克隆了一个响应干旱胁迫的基因ZmDi19-7，过量表达该基因的转基因拟南芥抗旱性显著增强。为了明确ZmDi19-7基因的生物学功能，本项目组获得了ZmDi19-7基因过量表达和CRISPR/Cas9敲除的转基因玉米种质材料，经PCR、测序、基因表达量分析等手段，检测出纯合过量表达和纯合敲除株系。然而，经过多次重复，本项目组发现过量表达ZmDi19-7基因的转基因株系和敲除突变体的耐旱能力与野生型均没有显著差别。不过，在多次实验过程中，本项目组发现ZmDi19-7基因敲除后，突变体苗期植株高度和生物量均显著高于野生型，而过量表达ZmDi19-7基因的转基因株系植株高度和生物量均显著低于野生型。此外，过量表达ZmDi19-7基因的转基因株系种子萌发速率和冷耐受性均显著降低，这暗示着ZmDi19-7基因能够负调控玉米苗期的生长发育和对冷害的耐受性，具有一定的实际应用价值。.为了解析ZmDi19-7负调控玉米苗期生长发育的分子机制，本项目利用转录组测序技术，分别将野生型与敲除突变体、过表达株系进行比较转录组分析，发现核糖体与蛋白质翻译等相关的通路显著富集，这暗示着ZmDi19-7基因可能会影响核糖体内蛋白翻译效率。此外，通过酵母双杂交实验，本项目组筛选了3个与ZmDi19-7互作的潜在靶标蛋白，即PSMC6、PSMD8以及ATP5。经功能注释发现，这三个蛋白分别编码26S蛋白酶体调控亚基T4、26S蛋白酶体调控亚基N12以及F-型氢离子转运ATP酶亚基O。通过BiFC、Split-LUC等实验，进一步验证了与ZmDi19-7蛋白之间的结合能力。研究还发现PSMC6、PSMD8蛋白的亚细胞定位与ZmDi19-7蛋白的分布高度一致，并且PSMC6、PSMD8之间也可以互作。本研究鉴定了ZmDi19-7的互作蛋白，阐明了互作蛋白与ZmDi19-7之间的互作关系，但这些互作蛋白是如何通过ZmDi19-7影响玉米生长发育的分子机制还有待进一步研究。