【作者】 杨利；

【导师】 赵术珍；

【作者基本信息】 山东师范大学 ， 植物学， 2021， 硕士

【摘要】 花生是我国重要的油料作物,主要分布在干旱和半干旱地区,水资源严重匮乏已成为限制花生产量的重要因素。干旱不但影响花生从萌发到结实的生长发育过程,而且严重影响花生的品质和产量,因此,研究花生的抗旱机理,对培育花生抗旱品种,提高花生产量和品质具有重要的指导意义。光是影响植物生长发育的重要环境因素。植物不仅依靠光合作用获取能量,还通过光受体感知光质、方向等信息,调节自身的生长发育。光敏色素phyA(phytochrome A)和phyB主要是远红光和红光受体,研究表明,phyA和phyB除了调控植物生长发育过程外,phyB还参与了植物对生物和非生物胁迫的响应,但是,对于phyA是否参与植物对逆境胁迫应答及其机理的研究,目前未见报道。本实验室在前期研究中获得了稳定的花生AhphyA过量表达株系(OEAhphyA),本研究以OEAhphyA和对照LH14为材料,在萌发期和苗期两个生长阶段,进行PEG胁迫和自然干旱胁迫(不浇水)两种处理,通过分析干旱胁迫后的生理指标及基因表达变化,揭示了花生AhphyA过量表达在干旱胁迫应答中的作用机理。主要研究结果如下:1.在种子萌发期,利用20%PEG胁迫处理,OEAhphyA三个株系6B4、6B9和6A12的发芽率分别为18%,38%和15%,显著低于对照LH14(93%),与对照LH14相比,OEAhphyA三个株系6B4、6B9和6A12的发芽指数均显著降低,到达萌发高峰期的时间也明显推迟,这说明AhphyA过量表达提高了花生种子萌发过程中对PEG胁迫的敏感性。2.选取苗龄10 d,长势一致的LH14和OEAhphyA幼苗,采用20%PEG溶液对其进行处理,与对照LH14相比,OEAhphyA株系叶片萎蔫程度较轻,植株形态较好。将LH14和6B4、6B9、6A12离体叶片置于实验室内恒温、避风、无阳光直射条件下自然风干,6B4、6B9和6A12的失水速率均显著低于对照LH14,说明OEAhphyA株系具有较高的保水能力,对干旱胁迫的耐受性较强。在胁迫条件下,OEAhphyA株系MDA含量显著低于对照LH14,CAT活性在20%PEG处理后均显著高于LH14。OEAhphyA株系的脯氨酸、可溶性糖和可溶性蛋白含量均显著高于对照LH14。OEAhphyA株系的叶片叶绿素荧光参数Fv’/Fm’、ФPSⅡ、qP、ETR和Fv/Fm较对照LH14均升高。这些结果说明,AhphyA过量表达提高了花生幼苗对PEG胁迫的耐受能力。3.将在正常浇水的土壤盆栽条件下播种25 d天且长势一致的LH14和OEAhphyA幼苗进行自然干旱胁迫处理,停止浇水16 d后发现OEAhphyA株系与对照LH14相比,叶片自然伸展,茎和叶片萎蔫程度均较轻。其MDA含量、渗透调节物质(脯氨酸,可溶性糖和可溶性蛋白含量)、Fv/Fm等叶片叶绿素荧光参数,在自然干旱胁迫后的变化与PEG处理变化一致,但SOD,POD和CAT抗氧化酶活性与PEG处理变化不同,这些结果进一步说明,AhphyA过量表达提高了花生幼苗的耐旱性。4.为了研究phyA提高花生耐旱性的机理,利用20%PEG胁迫处理的LH14和OEAhphyA株系幼苗为材料进行表达谱分析。结果表明,AhphyA过量表达引起与渗透调节物质、抗氧化酶相关基因及一些胁迫相关基因及转录因子在干旱胁迫前后表达变化显著。PEG胁迫处理下的差异表达基因主要与刺激响应、生物调节等生物学过程和分子功能,以及一些信号转导、糖类代谢等代谢途径有关。在20%PEG胁迫下,大量基因显著上调或下调,如endoglucanase 8-like、proline-rich protein 2-like、cyanogenic beta-glucosidase-like和beta-glucosidase12-like的编码基因,在20%PEG胁迫4 h后的表达水平显著上调。OEAhphyA株系中beta-amylase-like和sucrose synthase-like的编码基因,与对照LH14相比,其表达水平在20%PEG胁迫4h后显著下调。上述结果表明,AhphyA主要通过调节渗透调节物质相关基因的表达,增加渗透物质的积累,从而降低花生叶片失水速率,降低叶片膜脂受损程度,提高花生幼苗的光化学反应效率来提高花生对干旱胁迫的抗性。更多还原

【Abstract】 Peanut is an important oil crop in China,mainly distributed in arid and semi-arid areas.The severe shortage of water resources has become an important factor restricting peanut production.Drought affects not only the growth and development process of peanut from germination to maturing,but also seriously affect the quality and yield of peanut.Therefore,it is of significance to study the drought resistance mechanism for developing drought-resistant peanut varieties and improving peanut yield and quality.Light,an important environmental factor,affects the growth and development of plants.Plants not only rely on photosynthesis to obtain energy,but also perceive light quality and direction through photoreceptors to regulate their growth and development.Phytochrome A(phyA)and phyB are mainly red and far-red light receptors,which involve in the important process of plant growth and development.Furthermore,phyB also play a key role in biotic and abiotic stresses in plants.However,the roles and mechanisms of phyA involved in plant response to stress are not known.Stable peanut phyA overexpression lines(OEAhphyA)were obtained in the previous study.In this study,20% PEG treatment and natural drought stress(restrict watering)were conducted during seed germination and seedling stage of OEAhphyA and LH14 to investigate the mechanism of peanut phyA overexpression in response to drought stress.The main results are as follows:1.During the seed germination period,after 20% PEG treatment the germination rates of three OEAhphyA lines(as 6B4,6B9 and 6A12)were 18%,38% and 15%,respectively,which were significantly lower than the control LH14(93%).The germination index of the OEAhphyA lines(6B4,6B9 and 6A12)were remarkablely reduced to compare with LH14,and the time to reach the germination peak was also significantly delayed.The results indicated that the overexpression of AhphyA increased sensitivity to PEG treatment during peanut seed germination.2.LH14 and OEAhphyA seedlings of 10 d were treated with 20% PEG.The OEAhphyA lines treated with 20% PEG for 2 d showed a lighter wilting degree to compare with the control LH14.The detached leaves of LH14 and OEAhphyA lines were placed in the laboratory for drying under constant temperature,protected from wind direct sunlight.The water loss rates of the OEAhphyA lines(6B4,6B9 and 6A12) were significantly lower than that of LH14.OEAhphyA lines had higher water retention capacity,and higher tolerance to drought stress to compare with LH14.After 20% PEG treatment,the MDA contents of the OEAhphyA lines were significantly lower than that of LH14,and the CAT activity was significantly higher than that of LH14.The contents of proline,soluble sugar,and soluble protein of the OEAhphyA lines were significantly increased.The soluble protein content was significantly higher than that of the control LH14.The leaf chlorophyll fluorescence parameters Fv’/Fm’,ФPSⅡ,qP,ETR and Fv/Fm of the OEAhphyA lines were all higher than those of LH14.These results indicated that the overexpression of AhphyA increased the tolerance of peanut seedlings to PEG treatment.3.The 25 DAP (days of planting) LH14 and OEAhphyA seedlings,grown under the normally watered soil potting conditions,were subjected to natural drought stress.After stopping watering for 16 d,it was found that the leaves of the OEAhphyA lines were naturally stretched and the stems and leaves were less wilted to compare with LH14.The change of MDA content,osmotic adjustment substances(proline,soluble sugar and soluble protein content),Fv/Fm and other leaf chlorophyll fluorescence parameters after drought stress and PEG treatment were consistent.However,SOD,POD and CAT antioxidant enzyme activities,changes after drought stress and PEG treatment were inconsistent.These results further indicated that the overexpression of AhphyA improved the drought tolerance of peanut seedlings.4.In order to study the mechanism of phyA improving the drought tolerance of peanut seedlings,20% PEG treated seedlings of LH14 and OEAhphyA lines used for gene expression analysis.The expression of genes involved in osmotic regulation,antioxidant enzymes and stress-related genes and transcription factors changed significantly after 20% PEG treatment.The differentially expressed genes under PEG treatment were mainly related to stimulus response,biological regulation and other biological processes and molecular functions,some signal transduction,carbohydrate metabolism and other metabolic pathways.After 20% PEG treatment for 4h,the expression levels of endoglucanase 8-like gene,proline-rich protein 2-like gene,cyanogenic beta-glucosidase-like gene,beta-glucosidase 12-like gene were significantly up-regulated.In OEAhphyA lines,the expression levels of beta-amylase-like gene and sucrose synthase-like gene were significantly down-regulated after 20% PEG treatment for 4 h.These results indicated that AhphyA improved the resistance of peanut to drought stress by regulating the expression of osmotic regulation-related genes and increasing the accumulation of osmotic substances,thus reducing the water loss rate of peanut leaves,reducing the damage degree of leaf membrane lipid,and improving the photochemical reaction efficiency of peanut seedlings.更多还原