马尾松苗期应答低磷胁迫的分子机理研究

Pinus massoniana is the most important conifer in China, however, the phosphorous deficiency severely prohibited the growth of this tree. Previously, we had obtained some elite genotypes which showed significantly higher tolerance to low-phosphorous. To unravel the molecular mechanism of the tolerance to this stress, the present program will focus on distinguishing the differentially expressed genes and proteins under low-phosphorous stress using the strategy of next-generation sequencing technology and 2-D technoledge. Consequently, the chief candidate genes closely related to low-phosphorous stress of P. massoniana will be dug up, and the full length of low-phosphorous-related genes may be cloned and processed functional verification. Therefore, the ongoing project may elucidate the molecular mechanism underlying the elevated tolerance to low-phosphorous stress in low-phosphorous resistance germplasm, the molecular information and cloned genes can also provide a substantial platform for the genetic breeding for low-phosphorous tolerance in this tree.

马尾松是我国最主要优质针叶用材树种。本项目拟采用前期发掘的速生、耐低磷胁迫的马尾松种质为研究对象，通过转录组文库的高通量测序技术，获得低磷胁迫下差异表达的EST（Expressed Sequence Tag），根据生物信息学分析，筛选可能的耐低磷相关基因；另通过二维凝胶电泳和质谱技术，发掘低磷胁迫下的差异蛋白，探究耐低磷相关基因功能；通过相关基因的时空表达分析，建立耐低磷基因的表达谱；克隆耐低磷重要相关基因的全长序列，通过基因的超量及沉默（RNAi）表达，进一步验证其功能；结合低磷胁迫下其生理生化、转录组、蛋白组等研究结果，阐明马尾松苗期应答低磷胁迫的分子机理。项目开展，能为深刻认识马尾松耐低磷胁迫的机理提供信息，具有重要理论意义；耐低磷相关基因克隆，还为新的马尾松耐低磷资源的筛选及通过遗传工程创造耐低磷新种质奠定基础，具有一定实际意义。

马尾松是原产于我国南方地区的针叶树种，因其被广泛的用于木材、纸浆和树脂生产，已经成为我国最重要的经济树种之一。马尾松种植区土壤有效磷缺乏，严重制约了马尾松生长及经济产量。因此，探明马尾松耐低磷的分子调控机制，寻找低磷胁迫响应的关键基因，对筛选和培育磷高效吸收、利用的马尾松新种质，具有重要意义。本项目以前期筛选鉴定的耐低磷马尾松种质为材料，首先利用开发的马尾松IRAP标记筛选出遗传背景相对一致的幼苗，通过转录组文库的高通量测序获得低磷胁迫与正常供磷下马尾松的差异表达unigenes，根据其功能筛选到可能的耐低磷相关unigenes，建立了马尾松应答低磷胁迫相关基因的表达谱；另通过二维凝胶电泳和质谱技术发掘低磷胁迫下的差异表达蛋白，对差异蛋白点进行鉴定和功能分类；根据转录组和蛋白组结果，获得了显著差异表达的WRKY及MYB转录因子Unigene，并对其在重度低磷胁迫下时空差异表达进行分析；利用荧光定量PCR对低磷胁迫响应WRKY转录因子进行筛选，采用RACE方法克隆了应答低磷胁迫的WRKY转录因子（PmWRKY164）全长序列，构建了表达载体并进行遗传转化。以上结果为深入理解马尾松对磷吸收和利用的机理提供新信息，具有重要的理论价值；耐低磷相关基因的挖掘和克隆，还将为马尾松耐低磷新种质的筛选，以及通过遗传工程创造耐低磷新种质奠定基础，具有一定的实际意义。