马尾松高抗旱家系应答干旱胁迫的分子机理

Pinus massoniana is the most important conifer industrial timber in China, however, the drought severely prohibited the fast-growing and high-yield of this tree. Fortunately, we had obtained an elite genotype which showed significantly higher tolerance to drought. To unravel the molecular mechanism to this stress, the present program will focus on distinguishing the differentially expressed genes under drought stress using the strategy of next-generation sequencing and cDNA microarray technology, followed by the temporal and spatial expression profile elucidation. Consequently, the chief candidate genes closely related to drought stress of pine will be dug up, and the full length of drought-related genes may be cloned. Therefore, the ongoing project may elucidate the molecular mechanism underlying the elevated tolerance to drought stress in this germplasm, the molecular information and cloned genes can also provide a substantial platform for the genetic breeding for drought tolerance in this tree, as well as for further utility of this elite germplasm.

马尾松是我国最主要工业用材树种之一。项目拟采用自己选育的既速生丰产，又对干旱胁迫高抗的种质资源作为研究材料，利用转录组文库的高通量测序与cDNA Microarray技术相结合策略，获得干旱胁迫与正常水分下的差异表达基因，克隆到差异表达的EST（Expressed Sequence Tag），根据其功能筛选可能的抗旱相关EST；揭示马尾松在干旱胁迫下抗旱相关EST的时空表达，结合相关酶活性变化，建立干旱胁迫下抗旱基因的表达谱，在转录水平上解析马尾松应答干旱胁迫机制；最终筛选和克隆在抗旱中起重要作用的抗旱相关基因的全长序列。项目开展，不仅在转录水平解析马尾松高干旱种质抗旱特性的分子机制，也将为抗旱相关基因克隆和新的马尾松抗旱资源的筛选， 以及通过遗传工程创造抗旱新种质奠定基础；同时，项目也能直接为合理利用这一资源提供新思路，为全面准确的应用该良种打下坚实的理论基础。

干旱是影响植物正常生长发育的重要逆境因子之一，直接影响林木正常代谢，导致植物细胞脱水，膜、酶系统遭到破坏，细胞功能丧失，进而导致林木代谢紊乱，影响林木产量和品质。因此，通过分子生物学手段研究马尾松抗旱基因，探讨与揭示林木抗旱机理和认识植物抗旱本质，进一步提高林木抗旱能力成为现代林木研究工作中急需解决的关键问题之一。. 本项目采用本课题组选育的既速生丰产，又对干旱胁迫高抗的马尾松83号家系为研究材料，利用转录组文库的高通量测序技术，获得三个不同干旱胁迫时期与正常水分下的差异表达基因，克隆到差异表达的EST，建立了马尾松应答干旱胁迫相关基因表达谱；不同干旱时期共有的差异表达基因共有687个，主要参与了新陈代谢过程（如磷酸酶、蛋白激酶、合成酶、水解酶、氧化酶等）、脂代谢过程及氧化还原过程（如细胞色素P450、抗坏血酸盐氧化酶等）；GO 富集分析共获得374个GO功能注释，其中上调表达的Unigenes得到113个GO注释，下调表达的有261个GO注释；通过qRT-PCR验证及对功能基因比较分析后，获得了一批马尾松重要抗旱相关候选基因，并克隆其全长序列；构建了pBI121-PmPIP1、pSH737- PmCBL3、pBI121- PmGPX6、pBI121-PmPOD、pBI121-PmDHN 5个植物表达载体，转化至拟南芥进行过表达分析，结果表明，转基因植株较野生型植株的抗旱能力有所提升。