基于FOX捕捉系统挖掘盐生植物—沟叶结缕草抗盐基因的研究

The total area of saline area is 500 million mu in China, in which only one third were developed due to the serious effect of saline on the growth and development of plant. The breeding and utilization of salt tolerant plant is the direct and key measurement for the development of saline region.For accelerating progress of plant salt tolerance improvement,the explanation of molecular mechanism on salt tolerance and excavation of key salt tolerance genes on halophytes are current urgent problems.Halophyte-Zoysia matrella as a excellent perennial warm season turfgrass,is the important plant for improvement and landscape construction of saline soil. Previous research mainly focused on the salt tolerance evaluation and physiological mechanisms of Zoysia matrella, wheras its molecular mechanism of salt tolerance is still unclear. Therefore, extreme salt tolerance halophyte-Zoysia matrella Z123 selected from previous evaluation analysis on salt tolerance, is used for excellent salt tolerance gene mining from full-length cDNA expression library with cost-effective FOX hunting system. These salt tolerance genes are furtherly analyzed for molecular mechanism exploration based on bioinformatics,subcellular localization and fluorescent quantitative expression analysis. The implementation of this project, not only enrich the theory mechanism on molecular regulation of halophytes, and simultaneously provide excellent salt tolerance genes with independent intellectual property rights to speed up the process of salt tolerance breeding of new varieties.

我国盐碱土的总面积约有5亿多亩，由于盐胁迫严重影响了植物生长和产量，还有3亿多亩盐荒地无法开垦利用，而抗盐植物的选育与利用是提高盐渍土壤生产力和利用率直接而有力的措施。为了加快抗盐资源的育种进程，明确盐生植物的抗盐分子机制并找到关键的抗盐基因是目前急需解决的问题。盐生植物-沟叶结缕草作为优异的多年生暖季型草坪草，是盐渍地改良和绿化的重要资源。前期的研究主要集中在抗盐评价和生理机制方面，其抗盐分子机制尚不清楚。因此，本研究拟以前期筛选的极端抗盐盐生植物-沟叶结缕草Z123为材料，采用经济高效的FOX捕捉系统，从沟叶结缕草Z123盐诱导全长cDNA表达文库中挖掘优异的抗盐基因，并结合生物信息学、亚细胞定位、荧光定量表达分析，阐述其抗盐基因的分子调控机制。本项目的实施，不仅丰富了盐生植物抗盐分子调控机制，同时为植物抗盐遗传改良提供自主知识产权的优异抗盐基因储备，加快抗盐新品种选育的进程。

沟叶结缕草作为优异的多年生暖季型草坪草，是盐渍地改良和绿化的重要资源；以往的研究大多集中在抗盐评价和生理机制方面，其抗盐分子机制尚不明晰。本研究以前期筛选的极端抗盐沟叶结缕草Z123 为材料，通过磁珠抗体吸附、Gateway重组等技术成功构建了其全长cDNA植物表达文库，采用经济高效的 FOX 捕捉系统，创制了转cDNA文库基因的拟南芥种子库，经抗盐筛选和PCR扩增，初步获得了15个抗盐候选基因（ZmSAP8，ZmASR，ZmDUF1644，ZmGnTL，ZmSANT，ZmC2H2，ZmGRP，ZmLEA，ZmZAT，ZmLectin，ZmDBTNBT，ZmGRX，ZmUBP，ZmUAM和ZmVP1）。结合定量表达分析，进一步探索了其中3个受盐诱导上调表达的候选基因的耐盐机制，研究发现，（1）ZmVP1通过建立细胞质与液泡间的H+电势差，驱动Na+转运到液泡中，减轻细胞质内Na+的积累，维持较高的K+含量，同时，ZmVP1过量表达能促进NHX1、LEA、P5CS、SOD和APX等基因的表达；（2）ZmDUF1644过量表达显著上调了拟南芥离子转运相关基因（AKT1、NHX1和VP1），降低了拟南芥叶片中Na+积累，提高了K+离子含量，维持了更高的K/Na比；ZmDUF1644也促进了SOD、POD和APX的表达，提高了其酶活性，降低了盐胁迫下活性氧的积累；ZmDUF1644介导了根系和叶片中P5CS的上调表达，促进了脯氨酸积累；（3）过量表达ZmGnTL基因显著诱导了根系K+转运体（AKT1）和NHX1、SOD、APX、P5CS和BADH的表达，抑制PDH表达；进而缓解了盐胁迫下的离子毒害、氧化渗透胁迫，提高植物抗盐性。本研究的顺利完成，不仅获得了一些新型的抗盐基因，为草坪草抗盐分子育种提供了优异的靶标，同时也为进一步解析沟叶结缕草的抗盐分子机制奠定了基础。