【作者】 李健；

【导师】 杨兴洪；

【作者基本信息】 山东农业大学 ， 植物学， 2018， 硕士

【摘要】 Solanum tuberosum（简称St）型马铃薯是常见的栽培型马铃薯,然而St型马铃薯是冷冻敏感型品种且不具备冷驯化能力,栽培过程中经常遭受低温、干旱等非生物胁迫的损伤,严重影响马铃薯的产量和质量。Solanum commersonii（简称Sc）型马铃薯是一种野生型品种,具有较强的抗冻和冷驯化能力。CBF/DREB转录因子在植物应答低温、干旱等多种非生物胁迫中发挥着核心作用。为研究St和Sc型马铃薯的抗冻性差异是否与CBF1转录因子有关,我们将StCBF1和ScCBF1基因分别转入栽培型马铃薯（Solanum tuberosum）中,获得超表达CBF1基因的转基因植株。本研究选用野生型和CBF1基因表达水平相似的转基因马铃薯进行后续的实验。通过观察正常和胁迫条件下植株的生长表型,测定相关生理和生化指标,进而分析野生型和转基因马铃薯的抗逆性差异。主要结果如下:（1）马铃薯中超表达CBF1基因影响植株的生长和发育。其中超表达ScCBF1基因仅对植株生长早期产生一定的不利影响,薯块产量略有降低。然而超表达StCBF1基因却造成严重的生长迟滞现象,且薯块产量显著降低。（2）超表达ScCBF1与StCBF1基因均能增强马铃薯抗冻性,但ScCBF1植株的抗冻性显著强于StCBF1植株。转基因植株尤其是ScCBF1植株在低温胁迫下能够保持较高的膜脂不饱和程度,较低的电解质外渗率,较低的细胞膜氧化程度。LT₅₀实验表明ScCBF1植株具有最强抗冻能力,其LT₅₀为-4.5℃;StCBF1植株的抗冻性略有增强,其LT₅₀为-3.2℃;WT植株的LT₅₀为-2.5℃。（3）与野生型相比,转基因植株的抗旱性均显著增强;此外ScCBF1植株还表现出较强的抗盐能力。干旱胁迫后,转基因植株尤其是ScCBF1植株的失水速率和MDA含量显著低于野生型植株。表型分析发现,野生型植株叶片最早出现萎蔫表型,随后是StCBF1植株,ScCBF1植株最晚出现萎蔫表型。200mM·L^-1盐胁迫条件下,只有ScCBF1植株能够生根、生长。NMT测试结果显示:在盐胁迫条件下,WT与StCBF1植株根尖Na⁺、K⁺运输的动态平衡被打破,K⁺/Na⁺比降低;胁迫处理后ScCBF1植株的Na⁺外排增加,K⁺的吸收增加,从而能够维持细胞内较高的K⁺/Na⁺比,减轻Na⁺对细胞的毒害。（4）低温或干旱胁迫条件下,与野生型植株相比,转基因植株尤其是ScCBF1植株具有较高的叶绿素含量和气孔导度,因此能够维持较高的光合速率。胁迫处理后所有株系PSII的叶绿素荧光均发生变化,转基因植株尤其是ScCBF1植株的叶绿素荧光参数如V_I、V_J、ABS/RC、TR_O/RC及DI_O/RC均显著低于野生型;然而Fv/Fm和ET_O/RC却显著高于野生型。这表明在胁迫条件下转基因植株能够维持较高的能量利用效率,降低热耗散,从而维持较高的PSII活性。（5）正常或胁迫条件下,转基因植株尤其是ScCBF1植株能够维持较高的APX、POD、CAT和SOD活性;减少植物体内超氧阴离子和过氧化氢的积累,从而减轻活性氧对膜和光合系统的损伤。（6）ScCBF1和StCBF1转录因子能够影响诸多胁迫和代谢相关基因的表达。它们不仅能够直接调控COR基因的表达,而且还能够影响其它代谢过程相关基因的表达,如脂肪酸代谢相关基因、抗氧化酶相关基因、光合关键酶相关基因等。更多还原

【Abstract】 Solanum tuberosum（short for St）is a common cultivated potato,however,St-type potato is a freezing-sensitive variety and can not be cold-acclimated.In the cultivation process,it often suffers various abiotic stresses such as low temperature and drought.Solanum commersonii（short for Sc）is a wild-type potato with strong antifreeze and cold acclimation ability.CBF/DREB transcription factors play a key role in response to various abiotic stresses such as freezing and drought.In order to study whether the difference in frost resistance of St-and Sc-type potato is related to CBF1 transcription factor,we transferred StCBF1 and ScCBF1 genes to wild-type potato to obtain plants that overexpressing CBF1 gene.In this study,wild type and transgenic lines which have similar gene expression levels were used for subsequent experiments.By observing the growth phenotype of the plants under normal or stress conditions and measuring the relevant physiological and biochemical indicators,the differences in stress resistance between wild-type and transgenic plants were analyzed.The main results are as follows:（1）Overexpression of CBF1 gene in potato affected plant growth and development.The overexpression of ScCBF1 gene only had a little adverse effect on the early stage of plant growth,and the yield of tubers decreased slightly.However,overexpression of StCBF1 gene resulted in severe growth retardation and significantly decreased tuber yield.（2）Overexpression of ScCBF1/StCBF1 genes could enhance the freezing resistance of potato,but the freezing resistance of ScCBF1 plants was stronger than that of StCBF1 plants.Transgenic plants,especially ScCBF1 plants,can maintain higher membrane lipid unsaturation,lower electrolyte leakage,and lower cell membrane oxidation under cold stress.LT₅₀ experiments showed that the ScCBF1 plants had the strongest antifreeze ability,their LT₅₀0 was-4.5°C;the frost resistance of StCBF1 plants was slightly enhanced,their LT₅₀ was-3.2°C;the LT₅₀ of WT plants was-2.5°C.（3）Compared with the wild type,the drought resistance of the transgenic plants were significantly enhanced.In addition,the ScCBF1 plants also showed strong salt resistance.After drought stress,the water loss rate and MDA content of transgenic plants,especially ScCBF1 plants,were significantly lower than those of wild-type plants.Phenotypic analysis revealed that the leaves of wild-type plants first appeared a wilting phenotype,followed by StCBF1 plants,and the ScCBF1 plants showed a wilting phenotype at the latest.Under 200mM·L^-1salt stress,only ScCBF1 plants could root and grow.The results of NMT test showed that the dynamic balance of Na⁺and K⁺transportation in root tip of WT and StCBF1 plants was disrupted and K⁺/Na⁺decreased under salt stress,K⁺/Na⁺ratio decreased.After stress treatment,the Na⁺efflux of ScCBF1 plants increased and the K⁺uptake increased,which could maintain the higher K⁺/Na⁺ratio in the cells and reduce the toxicity of Na⁺to cells.（4）Under low temperature or drought stress conditions,the transgenic plants,especially ScCBF1 plants,had higher chlorophyll content and Gs compared to the wild type plants,and therefore could maintain a higher Pn.The PSII chlorophyll fluorescence of all lines were changed after stress treatment.The chlorophyll fluorescence parameters of transgenic plants,such as V_I,V_J,ABS/RC,TR_O/RC and DI_O/RC were significantly lower than that of wild type,however,Fv/Fm and ET_O/RC were significantly higher than WT plants.This indicates that under stress conditions,transgenic plants can maintain higher energy efficiency,reduce energy dissipation and maintain higher PSII activity.（5）Under normal or stress conditions,transgenic plants,especially ScCBF1 plants,can maintain higher APX,POD,CAT and SOD activity;reduce superoxide anion and hydrogen peroxide accumulation in plants,thereby reducing the damage of active oxygen on the membrane and photosynthetic system.（6）ScCBF1 and StCBF1 transcription factors can affect the expression of many stress and metabolism-related genes.They can not only directly regulate the expression of COR gene,but also can affect the expression of other metabolic processes related genes,such as fatty acid metabolism-related genes,antioxidant enzyme-related genes,photosynthesis key enzyme-related genes.更多还原