【作者】 周陈力；

【导师】 诸葛强；

【作者基本信息】 南京林业大学 ， 遗传学， 2010， 硕士

【摘要】 非生物胁迫如干旱、极端温度和盐胁迫,严重影响林木生长和产量。我国有三亿亩属于盐碱地,且干旱面积在不断增加,所以如何解决非生物胁迫是林木植物研究迫切需要解决的重要问题。AtGolS2基因(肌醇半乳糖苷酶基因)是拟南芥中GolS基因家族中的一员,受干旱和盐胁迫诱导。SRK2C基因是SnRK基因家族中的一员,SnRK(蔗糖非发酵相关蛋白激酶)通过多种复杂的信号传递途径对干旱、高盐、低温胁迫作出响应,激发抗逆基因表达,提高植物的抗逆性。本研究以南林895杨为材料,运用农杆菌介导法,开展了抗逆相关基因AtGolS2基因和SRK2C基因的遗传转化研究,主要研究结果如下:1.遗传转化中选择压的确定。本研究采用的筛选标记基因是潮霉素磷酸转移酶(hpt)基因,在遗传转化中采用潮霉素作为筛选转化植株的选择剂。经过分析比对试验,结果表明采用Hyg2.5mg/L作为筛选时的选择压。2.遗传转化体系的优化:分析比较了影响农杆菌转化的多个因素(预培养时间、菌液浓度、侵染时间、共培养时间等),获得杨树合适的遗传优转化条件:预培养3d,菌液浓度OD6000.8左右,侵染时间15～20min,共培养时间4d。3.通过遗传转化条件的优化及潮霉素筛选获得了一批转AtGolS2基因和SRK2C基因的南林895杨植株,并对转基因植株进行了分子生物学鉴定和耐盐性试验。4.分子生物学鉴定(包括PCR和RT-PCR分析)结果显示目的基因已经整合进杨树基因组中。耐盐性初步试验表明转基因植株耐盐性有一定提高。更多还原

【Abstract】 Abiotic stresses such as drought,extreme temperature,salt injury which can affect the growth and quantity of forest trees sharply.In China,there are three million hectares are saline land,and arid area is increasing.So it is urgent to solve the abiotic stresses in forest research.At least seven GolS-related genes are found in the Arabidopsis genome.AtGolS2 is a member of GolS gene family in Arabidopsis thaliana.AtGolS2 were induced by drough and high-salinity stresses,but not by cold stress.SRK2C gene is a member of SnRK gene family.SnRK genes are capable of mediating signals initiated during drought,salt and cold stress,play important roles in transducing stress signals,increasing protein synthesization and regulating signal transduction.In this study,Populus×euramericana cv.‘Nanlin895’was used as experiment material,adapting to Agrobacterium-mediated to introduce separately two resistance gene of AtGolS2 gene and SRK2C gene.The main conclusion of this experiment was as following:1.Determination of selective pressure of transformation:the selectable marker gene of AtGolS2 and SRK2C were both htp , so adopting Hyg as selective agent in transformation.Through contrast experiment,it is 2.5mg/L Hyg as selective pressure.2.Establishment of the efficient system of transformation:we studied several factors affecting Agrobacterium-mediated transformation,drawing the best transform condition: pre-culture time were 3 days, bacterial concentration OD600 0.8,infection time were 15～20 minute,coculture time were 4 days.3.Transgenic poplars were selected to survive on the culture medium with antibiotics,which includes Populus×euramericana cv.‘Nanlin895’with single AtGolS2 gene and SRK2C gene.The molecular biological tests and salt stress experiment were carried out to verity these transgenic poplar.4.The two genes had been integrated into poplar genome and could express,which verified by PCR and RT-PCR.The result of salt treatment showed that salt-resistance ability of transgenic poplars with single AtGolS2 gene and SRK2C gene were enhanced.更多还原