【作者】 王丹；

【导师】 国静；

【作者基本信息】 山东农业大学 ， 林业（专业学位）， 2020， 硕士

【摘要】 由于人类活动和全球气候变化,越来越多的土地出现盐渍问题,给农林业发展带来日渐严重的损失。盐胁迫会对植物造成离子失衡、渗透胁迫与氧化胁迫等伤害,导致植物生物量下降,甚至导致植物的凋零死亡。因此,研究植物耐盐机理,提高植物的耐盐能力对于提高林业生产力具有重要意义。我国的杨树人工林面积居世界第一位,杨树已成为解决我国木材供应的一个重要树种,近年来杨树作为林木基因工程的模式树种得到广泛研究。本课题组在前期研究中已获得了组成型过表达MPK7基因的‘84K杨’转基因植株(P_35S::MPK7^OE)。本研究采用水培系统,研究P_35S::MPK7^OE转基因杨树幼苗在不同浓度NaCl胁迫下和不同胁迫时间下各项生理生化指标的变化。研究结果如下:（1）高盐胁迫明显抑制杨树叶片表型。在NaCl浓度为0¹00 mM时,杨树叶片没有出现明显表型变化;当用150 mM NaCl处理时,对照幼苗受到严重胁迫,其叶片失水萎蔫,而OE 3-3、OE 1-6转基因杨树幼苗受胁迫程度明显低于对照植株,且表达量高的株系受胁迫程度较低,表明过表达PeMPK7转基因杨树的耐盐能力优于对照植株。（2）不同浓度的NaCl胁迫显著降低杨树光合作用能力。随着NaCl浓度的升高,杨树的光合速率（P_n）、蒸腾速率（T_r）、气孔导度（G_s）及水分利用效率（WUE）等参数明显降低,过表达PeMPK7转基因杨树P_n的下降幅度明显低于对照植株;表明PeMPK7在一定程度上缓解了NaCl胁迫对光合速率的抑制,减轻了盐胁迫对光合系统的损伤。在NaCl浓度为50 mM时,P_n降低,C_i降低,L_s上升,NaCl浓度大于50 mM时,C_i升高,L_s降低,表明NaCl浓度为50 mM是光合速率降低由气孔限制转向非气孔限制的转折浓度。（3）不同浓度的NaCl胁迫显著降低杨树光化学效率和电子传递效率。随着NaCl浓度的增大,PeMPK7转基因杨树和对照植株变化趋势基本一致,最大荧光（F_m）、实际光化学效率(Φ_PSⅡ)、最大光化学效率（F_v/F_m）、潜在光化学效率（F_v/F_o）及光化学猝灭（qP）均明显降低,最小荧光（F_o）及非光化学猝灭（NPQ）显著升高,表明盐胁迫导致杨树发生光抑制现象;而PeMPK7转基因杨树的变化幅度优于对照植株。以上表明,过表达植株能缓解高浓度NaCl胁迫导致的光抑制、PSⅡ反应中心的关闭和电子传递效率低等问题,且表达量高的植株受到的缓解程度较明显。（4）NaCl浓度胁迫及胁迫时间导致杨树出现氧化胁迫和渗透胁迫。随着NaCl浓度及胁迫时间延长,丙二醛（MDA）含量增高,此时植物体内会通过提高可溶性糖、可溶性蛋白的含量和抗氧化酶的活性来抵抗氧化胁迫和渗透胁迫;PeMPK7转基因杨树和对照植株变化趋势基本一致,但PeMPK7转基因杨树MDA含量上升幅度低于对照植株,表明转基因杨树膜损伤程度轻于对照,PeMPK7对于膜功能的维护上具有一定作用,且表达量越高对细胞膜的保护作用越大;而可溶性糖和可溶性蛋白的含量和抗氧化酶的活性上升幅度明显高于对照植株,且PeMPK7基因表达量越高,可溶性糖和可溶性蛋白的含量和抗氧化酶的活性越高,表明PeMPK7在一定程度上通过维持转基因杨树的渗透调节,增强对活性氧的清除能力,提高‘84K杨’的耐盐性,且表达量越高抗氧化酶活性越强。更多还原

【Abstract】 Due to human activities and global climate change,more and more land appears salinization problem,which brings increasing serious losses to the development of agricultural and forestry.Salt stress can cause damage to plants,such as ion imbalance,osmotic stress and oxidative stress,which leads to the decline of plant biomass and even the death of plants.Therefore,it is of great significance to study the mechanism of plant salt tolerance and improve the ability of plant salt tolerance to improve forestry productivity.The area of Poplar plantation in Chinaranks the first in the world,and Poplar has become an important tree species to solve the problem of wood supply in China.In recent years,as a model tree species of forest genetic engineering,poplar has been widely studied.In the previous study,the’84K Yang’transgenic plantswith the overexpression of MPK7 gene(P_35S::MPK7^OE)have been obtained by our team.In this study,a hydroponic system was used to study the changes of physiological and biochemical indexes of P_35S::MPK7^OEE transgenic poplar seedlings under different concentrations of NaCl stress and different stress time.The results are as follows:（1）High salt stress significantly inhibited the leaf phenotype of Poplar.When NaCl concentration was 0¹00 mM,there was no significant phenotypic change in Poplar leaves.When NaCl concentration was 150 mM,the contrast seedlings were severely stressed,and the leaf wilted due to water loss,However,the stress level of OE 3-3and OE 1-6 transgenic poplar seedlings was significantly lower than that of the control plants,and with the amount of expression increasing,the stress phenotype was lower,showing that the salt-tolerant ability of transgenic poplar with overexpression of PeMPK7 was than that of the control plants.（2）Different concentrations of NaCl stress significantly reduced the photosynthetic capacity of Poplar.With the NaCl concentration increasing,the photosynthetic rate（P_n）,transpiration rate（T_r）,stomatal conductance（G_s）and water use efficiency（WUE）of poplar decreased significantly,and the decrease of P_n in transgenic Poplars with overexpression of PeMPK7 was significantly lower than that of control plants;indicating that PeMPK7alleviated the inhibition of NaCl stress on P_n and the damage of salt stress on photosynthetic system to a certain extent.When NaCl concentration was 50 mM,P_n decreased,C_i decreased and L_s increased;when NaCl concentration was greater than 50 mM,C_i increased and L_s decreased,indicating that NaCl concentration of 50 mM was the turning point concentration of photosynthetic rate from stomatal restriction to non-stomatal restriction.（3）The photochemical efficiency and electron transfer efficiency of Poplar were significantly reduced by different concentrations of NaCl stress.With the NaCl concentration increasing,the PeMPK7 transgenic poplars and control plants have almost the same change trend,namely the maximum fluorescence（F_m）,the actual photochemical efficiency(Φ_PSⅡ)and the maximal photochemical efficiency（F_v/F_m）,potential photochemical efficiency（F_v/F_o）and photochemical quenching（qP）were significantly lower,minimal fluorescence（F_o）and photochemical quenching（NPQ）increased significantly,indicating that salt stress led to the Poplar photoinhibition;The change range of PeMPK7 transgenic Poplar was better than that of control plants.Above showed that overexpression plants could alleviate the problems of light inhibition,closure of PSⅡreaction center and low electron transfer efficiency caused by high concentration of NaCl stress,and the higher the expression quantity,the more obvious the alleviate degree.（4）The concentration and duration time of NaCl stress led to oxidative stress and osmotic stress in Poplars.With the NaCl concentration and stress time increasing,malondialdehyde（MDA）content increased.At this time,the plant would resist oxidative stress and osmotic stress by increasing the content of soluble sugar,soluble protein and antioxidant enzyme activity.The change trend of PeMPK7 transgenic Poplar was basically the same as that of control plants,but the increase of MDA content in PeMPK7 transgenic Poplar was lower than that of control plants,indicating that the degree of membrane damage of transgenic Poplar was less than that of control plants,and the PeMPK7 played a certain role in the maintenance of membrane function,and the higher the expression level,the greater the protective effected on cell membrane.The content of soluble sugar,soluble protein and the activity of antioxidant enzymes increase significantly higher than that of control plants,and the higher the expression of PeMPK7 gene,the higher the content of soluble sugar and protein and the activity of antioxidant enzyme,indicating that PeMPK7 could maintain the osmotic regulation of genetically Poplar,inhance the ability of scavenging active oxygen,and improve the salt resistance of’84K Yang’to some extent,and the higher the expression level,the stronger activity of the antioxidant enzymes.更多还原