花生苗期响应干旱胁迫关键基因挖掘与功能鉴定

Peanut in seedling stage is very sensitive to drought, and it is the key limiting factors in spring for high and stable yield of peanut in Liaoning province. In the previous research, a pair of sister lines of drought-tolerant Nonghua5-1 (NH5-1) and drought-sensitive Nonghua5-2 (NH5-2) was screened by PEG stress and rainproof shelter experiments. Therefore, paraffin sections and scanning electron microscopy techniques were used to reveal cytological and spatiotemporal changes responding to drought stress of peanut at seedling stage. The transcriptome (RNA-Seq) sequencing technology was applied to explore differentially expressed genes, which was verified by RT-qPCR, in response to drought stress; and then differentially expressed proteins in response to drought stress were screened by IBT quantitative proteomics. It was association analysis that used to analyze differentially expressed genes in both transcriptome and proteome, and verification was fulfilled by molecular hybridization. Genetic transformation of arabidopsis or tobacco to verify gene function was performed by constructing over-expressed vector of candidate gene. The results can lay a foundation for revealing the molecular mechanism of peanut drought stress and provide a theoretical reference for peanut drought resistance breeding.

花生苗期对干旱非常敏感，春季苗期干旱是辽宁花生高产稳产的最大限制因素。前期课题组通过盆栽PEG胁迫和干旱棚控水试验，筛选出苗期耐旱型农花5-1（NH5-1）和干旱敏感型农花5-2（NH5-2）的一对姊妹系材料。在此基础上，利用石蜡切片和扫描电镜技术，揭示花生苗期应答干旱胁迫的细胞学时空变化规律；利用转录组（RNA-Seq）测序技术，挖掘响应干旱胁迫的差异表达基因，并对候选差异基因进行RT-qPCR验证；利用IBT定量蛋白质组学技术，筛选应答干旱胁迫的差异表达蛋白；关联分析转录组和蛋白质组共有差异表达基因，利用分子杂交技术进行验证；构建候选基因过表达载体遗传转化拟南芥或烟草验证基因功能。研究结果可为揭示花生干旱胁迫的分子机制奠定基础，为花生抗旱育种工作提供理论参考。

由于近年来气候的极端异常变化，导致辽宁花生产区春旱频发，严重限制了该区域花生安全生产。本项目通过探索不同类型花生品种（耐旱型花育22 HY22和敏感型阜花18 FH18）苗期应对干旱胁迫的形态、生理、组织细胞及基因表达差异，以期揭示二者响应干旱胁迫的差异特征。主要结果包括：20%PEG-6000干旱胁迫浓度下，HY22根系酶促抗氧化酶系统在胁迫9 h后开始启动，过氧化氢、单线态氧含量和脂质过氧化程度较低，根尖组织细胞结构较完整，细胞内部高尔基体、内质网和液泡结构清晰，细胞内含物减少，并能保持较高根系活力及侧根密度；FH18表现相反。HY22地上部分与根系表现类似，抗氧化系统启动较早且含量较高，过氧化氢、单线态氧含量及脂过氧化程度较低，渗透调节物质含量较高；叶片气孔胁迫12 h时关闭，且9 h时栅海比为1.33，茎部皱缩但髓部结构完整；叶片光合系统II光化学反应的效率和光合作用潜力较高，非光化学淬灭系数较低；而FH18气孔关闭发生在胁迫16 h，9 h栅海比为1.05，叶绿素荧光等指标变化均为相反趋势。表明耐旱品种HY22可通过快速激发抗氧化系统，清除细胞内过氧化氢等有毒物质保持其生存能力。RNA-seq发现HY22干旱胁迫后，根系中萜类骨架合成途径、ABA信号传导途径及GA生物合成途径中的关键酶基因均上调表达；HY18则相反。地上部分组织中亚麻酸代谢、光合作用天线蛋白、类黄酮生物合成和苯丙类生物合成途径差异较大，表明基因表达水平上HY22具有快速激发响应逆境胁迫的信号传递能力，进而提高自身的防御机能，表现出干旱胁迫生存的适应性优势。上述研究结果可为系统研究干旱胁迫对花生苗期各组织器官的影响，分析花生响应干旱胁迫差异的关键代谢通路，鉴定关键基因提供理论参考；同时可为筛选抗旱种质提供理论依据。