黄瓜CsNMAPK和CsPLDα共表达对盐胁迫的响应机制研究

Under the conditions of adversity,MAPK cascades signaling pathways and PLD signaling pathways could activate a variety of cell reactions to increase stress tolerance of plant. Does co-expression of MAPK and PLD in transgenic plant exist interactions effects? Study on that was virtually lacking. In our study , the regeneration system in cucumber (Cucumis sativus L.) and Agrobacterium tumefaciens mediated genetic transformation were constructed.We found that over-expression of CsNMAPK in transgenic cucumber could trigger a burst of NO and an increase of ABA synthesis, and reduce H2O2 content and antioxidant enzyme activities. The contents of MDA of inhibiting-expression lines were significant higher than wild type. However, SOD activity and Pro content were significantly lower than wild type, suggesting that CsNMAPK was related to positive regulation of ROS scavenger and osmoregulation under NaCl stress. Based on former study, co-expression of CsNMAPK and CsPLDα in transgenic cucumber could be obtained by Agrobacterium-mediated and flowering hybrid. Transgenic cucumbers were treated with salt under hydroponic cultivation. The transcriptional and translational expression of CsNMAPK and CsPLDα in leaves and roots of transgenic and wild type cucumber plants were analyzed by Southern Blot, Northern Blot and Western Blot. The amount change of endogenous level of NO, Hormones (ABA et al), JA and SA, and reactive oxygen species were also analyzed to study responses of co-expression of CsNMAPK and CsPLDα signaling pathways to NO3- stress. More emphasis was laid on responding mechanisms of co-expression of CsNMAPK and CsPLDα in transgenic cucumber to salt stress.

逆境胁迫下，植物通过活化的MAPK级联信号途径和PLD信号转导途径激活各种细胞反应，从而提高植物对逆境的抗性，那么两者共表达是否存在互作效应，至今尚未见报道。我们构建了农杆菌介导法获得转基因黄瓜植株的遗传转化体系；前期工作研究发现，NO3-胁迫下，CsNMAPK过表达引发了NO的猝发，增加了ABA的合成，降低了H2O2含量和抗氧化酶活性；NaCl胁迫下，抑制CsNMAPK表达黄瓜幼苗的MDA含量显著高于野生型，而SOD活性和Pro积累显著低于野生型。本课题在此基础上，拟采用农杆菌介导法和花期相互授粉分别得到CsNMAPK和CsPLDα共转化黄瓜植株，采用营养液培养模拟土壤盐渍化，从转录和翻译水平上分析比较盐胁迫下转基因和野生型黄瓜幼苗中CsNMAPK和CsPLDα的表达，结合内源信号分子、活性氧系统、膜磷脂组分和质子泵活性等，研究CsNMAPK和CsPLDα共表达对盐胁迫的响应机制。

（1）成功构建了pROKII- CsNMAPK（+）- CsPLDα（+）。.（2）利用农杆菌介导法分别获得转pBI-CsPLDα（+）、pBI-CsPLDα（-）和pROKII- CsNMAPK（+）- CsPLDα（+）黄瓜株系14、8和4个。.（3）盐胁迫下，黄瓜幼苗叶片中CsPLDα和CsNMAPK基因及蛋白表达量和酶活性分别在处理1h和3h后达最大；NO含量在1h后达最高，SOD和APX活性和PA含量在3h后达最高，CAT和POD活性在5h后较高，而H2O2和O2-含量随处理时间延长而增加。以上结果说明，CsPLDα和CsNMAPK基因对盐响应存在时间差，且CsPLDα响应早于CsNMAPK，初步推测此两个基因可能存在互作效应。.（4）正常生长条件下，P+和P+M+中CsPLDα表达量显著高于其他株系，而P+、M+和P+M+中CsNMAPK表达量亦显著高于其他株系。盐胁迫1h和3h后，P+、M+和P+M+中CsPLDα表达量在1h后达最大， CsNMAPK表达在3h后达最大，再次证明两者存在时间差。盐胁迫下，随时间延长所有转基因黄瓜株系中ABA、 JA和GA含量均有所升高，SA含量降低； M-中SOD和CAT活性升高，M+中POD活性升高，P+M+中APX活性升高，其他株系中SOD、CAT、POD和APX活性均呈降低趋势；P+、P-和P+M+中膜磷脂总DGDG和MDDG含量、P+和P-中PG含量、P-和P+M+中PC和PE含量均呈升高趋势，但其他株系中膜磷脂成分呈降低趋势或变化不明显，所有转基因株系中PA含量升高；WT中SA、JA、DGDG、PG、PE 、PS和PA含量降低，但GA、MGDG含量和抗氧化酶活性升高；PA中所有组分34:3、36:6 、36:5和36:3均有升高趋势，其他磷脂中组分亦有变化，但不同组分变化趋势不同。以上结果表明，CsPLDα介导的信号转导途径可启动下游CsNMAPK信号途径，初步表明CsPLDα和CsNMAPK对盐胁迫存在互作响应。.（5）盐和干旱胁迫下，转基因CsPLDα烟草幼苗具有较强的保水能力，且在一定程度上可减轻膜脂过氧化，表明CsPLDα基因参与了植物气孔、渗透调节力及活性氧清除的调控。.（6）短时间和长时间250mM NaCl 胁迫后，CsPLDα可通过维持离子平衡和活性氧系统平衡、积累渗透调节物质来提高耐盐性。