干旱、高盐胁迫耐受性相关的胡杨PeNAC基因的功能解析

NAM, ATAF1,2, and CUC2 (NAC) proteins have received considerable attention, due to their regulation function in many biological processes, including stress response, cell metabolism, and plant growth and development. To date, little is known about the stress-related NAC genes in poplar. In our previous study, we analyzed the differences of the transcriptomes between the transgenic poplar lines overexpressing PtMKK4 and the non-transgneic lines using RNA sequencing technique, and obtained a subset of NAC genes which were up-regulated in the transgenic lines. To judge whether this subset of NAC genes participated in poplar responses to stresses or not, we further compared the expression patterns of these NAC genes in Populus euphratica with those in Populus trichocarpa under diverse stress treatments. The results showed that two NAC genes of Populus euphratica (PeSNAC1 and PeSNAC2) were up-regulated obviously by drought or high salinity treatment, while the expression levels of their homologous genes in Populus trichocarpa showed little changes, indicating that PeSNAC1 and PeSNAC2 may play important roles in the response to drought or high salinity in Populus euphratica. With the help of the established research platforms such as poplar genetic transformation system, protoplast transient expression system, etc., the following research work will be carried out, which includes: determining the upstream signal regulating the expression of PeSNAC1 and PeSNAC2; comprehensively analyzing the functions of PeSNAC1 and PeSNAC2; identifying the downstream target genes and the interacting proteins of PeSNAC1 and PeSNAC2, and ascertaining their regulation pathways. Based on the data obtained, we will build the regulatory networks of PeSNAC1 and PeSNAC2 in Populus euphratica. Our study will provide insights into the molecular resistance mechanisms of Populus euphratica, and lay solid foundations for improving the poplar stress resistance via genetic engineering.

NAC转录因子因其广泛参与植物逆境应答、细胞代谢及生长发育过程而备受关注。迄今，关于杨树胁迫相关NAC功能及其作用机制的报道非常有限。前期研究中，本课题组从耐旱性增强的PtMKK4过表达转基因杨树中筛选到一组表达上调的NAC基因，进一步比较其在抗逆胡杨和非抗逆毛果杨中对逆境响应模式的差异，发现2个NAC基因（PeSNAC1和PeSNAC2）在胡杨中对干旱或高盐处理高水平应答，而其毛果杨对应同源基因的表达水平变化不明显，暗示其可能与胡杨对干旱、高盐的耐受性密切相关。本课题将确定调控PeSNAC1和PeSNAC2表达的上游信号；全面解析其生物学功能；鉴定其下游靶基因、结合位点及与其相互作用的蛋白质，探明其调控途径。在此基础上，对其中的重要节点进行鉴定、验证，建立PeSNAC1和PeSNAC2的基因调控网络。本项目的完成将为深入理解胡杨抗逆机理提供新资料，同时为杨树抗逆性遗传改良提供理论依据。

NAC转录因子因其广泛参与植物逆境应答、细胞代谢及生长发育过程而备受关注。本研究围绕从胡杨中筛选到的2个胁迫相关NAC基因（PeSNAC1和PeSNAC2）开展以下研究，以期为深入理解胡杨抗逆机理提供新资料，同时为杨树抗逆性遗传改良提供理论依据：.（1）全面分析胡杨PeSNAC1和PeSNAC2对各种非生物胁迫的响应模式。结果显示，PeSNAC1和PeSNAC2对NaCl、PEG、H2O2和ABA处理后表现不同的变化趋势，其中PeSNAC1在胁迫处理后的上调幅度更为显著。.（2）鉴定PeSNAC1和PeSNAC2的生物学功能。结果显示，PeSNAC1为定位于细胞核中的转录激活因子；PeSNAC2定位于细胞核中，但未检测到其转录激活活性。高盐胁迫处理下，过表达PeSNAC1的转基因山新杨和拟南芥的生长显著优于野生型。而过表达PeSNAC2转基因植株与野生型之间没有明显差异。.（3）筛选转基因植株和野生型之间差异表达的基因，寻找关键下游目标基因。对在转基因植株中显著上调的肌醇-1-磷酸合酶基因MIPS1进行功能鉴定。结果显示，转基因杨树对高盐和Cu胁迫的耐受性明显高于野生型。进一步研究结果表明，过表达MIPS1可增强转基因植株中AsA/GSH循环的速率提高其抗氧化能力，从而降低了胁迫引起的氧化伤害。.（5）将前期鉴定出的重要抗逆基因PeSNAC1和PeMIPS1应用于速生林‘欧美杨107’抗逆遗传改良。现已获得转基因‘欧美杨107’株系，目前正在对其表型进行鉴定分析，有望获得可在山东等地大面积推广的抗逆速生杨新品种，实现对大面积盐渍化地的高效利用。.（4）利用转录组测序，寻找、挖掘毛果杨木质部中对盐胁迫响应的关键调控因子。筛选到836个上调基因和 914个下调基因。值得关注的是，这些差异表达基因(DEGs)在糖代谢途径（Carbohydrate metabolism）富集程度最高，其次是在信号转导（Signal transduction）途径。挑取了2个PtLIMs和3个PtIQDs进行功能鉴定，通过初步表型分析发现过表达PtLIM1转基因山新杨茎中的纤维素含量明显多于野生型。其余的转基因植株的表型要进一步分析鉴定。