【作者】 王媛花；

【导师】 章镇；

【作者基本信息】 南京农业大学 ， 果树学， 2008， 硕士

【摘要】 草莓(Fragaria ananassa Duch)属于蔷薇科草莓属多年生草本植物,是一种重要的经济浆果,在世界范围内广泛种植。然而草莓安全越冬和耐寒品种的缺乏成为生产上的突出问题。生物技术的飞速发展为解决这一问题提供了新的途径。水稻转录因子RdreB1BI是一个调控植物发育且与植物胁迫抗性相关的转录因子。已有研究证明将RdreB1BI基因转化拟南芥,拟南芥植株的抗早性和耐寒性有很大提高。到目前为止还未见有RdreB1BI基因转化草莓的研究。因此本试验目的是用根癌农杆菌介导的转化方法将水稻RdreB1BI基因导入草莓细胞,获得抗寒的转基因植株。主要研究结果有以下几点:1.以草莓‘丰香’、‘雪蜜’、‘鬼怒甘’的叶片和叶柄为外植体,建立了高效离体再生体系。‘丰香’叶片和叶柄的最佳不定芽再生培养基为:MS+TDZ(2.0mg/L)+IBA(0.2mg/L)+2,4-D(0.1mg/L)和MS+B5+TDZ(2.0mg/L)+IBA(0.2mg/L)+2,4-D(0.1mg/L)。‘雪蜜’叶片和叶柄的最佳不定芽再生培养基为:MS+TDZ(1.5mg/L)+IBA(0.2mg/L)和MS+B5+TDZ(2.0mg/L)+IBA(0.2mg/L)。‘鬼怒甘’叶片和叶柄的最佳不定芽再生培养基为:MS+BA(2.0mg/L)+IBA(0.1mg/L)和MS+BA(2.0mg/L)+IBA(0.1mg/L);‘丰香’、‘雪蜜’暗培养时间为14天,‘鬼怒甘’暗培养时间为7天;‘丰香’、‘鬼怒甘’叶片不定芽再生的最佳叶龄为10-30天,‘雪蜜’叶片不定芽再生的最佳叶龄为20天以下。三个草莓品种叶柄不定芽再生的最佳叶龄都是20天以下;叶片放置方式对三个草莓品种的叶片不定芽再生没有影响,叶柄刻伤向上放置有利于三个草莓品种叶柄不定芽再生。2.克隆了rd29A启动子,通过对比分析,与已公布的序列有98.72%的同源性,主要调控区域也一致。构建了两个植物表达载体pYH4215-rd29A和pYF7713-Rdre。3.通过研究影响遗传转化的因素,建立了农杆菌介导的‘雪蜜’草莓遗传转化体系:菌液浓度为OD600=0.4;侵染时间为9min;选用200mg/L的Cb作为抑菌抗生素,20mg/L的Kan作为筛选抗生素;共培养时间3天;共培养培养基:MS+TDZ(2.0mg/L)+IBA(0.2 mg/L);筛选培养基:MS+TDZ(2.0mg/L)+IBA(0.2mg/L)+Kan(20mg/L)+Cb(200mg/L);伸长培养基:MS+6-BA(0.5mg/L)+NAA(0.1mg/L)+Kan(20mg/L)+Cb(200mg/L);生根培养基:MS+IBA(0.1mg/L)+Kan(20mg/L)+Cb(200mg/L)。4.获得了Kan抗性的含有RdreB1BI基因的草莓转基因株系15株,PCR以及RT-PCR检测证明,有4个株系外源基因已经成功的导入和整合到草莓基因组中。脯氨酸含量的测定和低温处理表明,转基因的植株较非转基因植株抗寒性有所提高。更多还原

【Abstract】 Strawberry(Fragaria ananassa Duch),perennial herbaceous plant in the Rosaceae family,which is a major berry crop around the world.However,safe hibernation and deficiency of cold tolerance varieties are outstanding problems.In this case,biotechnology which developed speedly is an alternative efficient strategy to solve the problems. RdreB1BI gene is important transcription factors which related to plant development and physiology.It was investigated that RdreB1BI transcription factors are able to improve tolerance to drought,high-salt and cold stresses in Arabidopsis.But there is no research in transformation of strawberry with RdreB1BI gene of rice at present.So in this study, RdreB1BI was used as purpose gene,the transformation of strawberry by Agrobacterium-mediated was studied.The experiment research results were as follows:1.The ideal adventitious shoot regeneration system of strawberry ’Toyonoka’,’Xuemi’ and ’Guinugan’ by leaf and petiole explants were established In this study.The results showed that the highest adventitious shoot regeneration rate of Toyonoka’s leaf disc was obtained on MS medium supplemented with 2.0mg/L TDZ,0.2 mg/L IBA and 0.1mg/L 2,4-D.The highest adventitious shoot regeneration rate of Toyonoka’s petiole was obtained on MS+B5 medium supplemented with 2.0mg/L TDZ,0.2mg/L IBA and 0.1mg/L 2,4-D. The highest adventitious shoot regeneration rate of Xuemi’s leaf disc was obtained on MS medium supplemented with 1.5mg/L TDZ and 0.2mg/L IBA.The highest adventitious shoot regeneration rate of Xuemi’s petiole was obtained on MS+B5 medium supplemented with 2.0 mg/L TDZ,0.2mg/L IBA.The highest adventitious shoot regeneration rate of Guinugan’s leaf disc was obtained on MS medium supplemented with 2.0mg/L BA and 0.1mg/L IBA.The highest adventitious shoot regeneration rate of Guinugan’s petiole was obtained on MS medium supplemented with 2.0 mg/L BA and 0.2mg/L IBA.For ’Toyonoka’ and ’Xuemi’ Dark treatment for two weeks could increase the adventitious shoot regeneration.But for ’Guinugan’ Dark treatment for one week could increase the adventitious shoot regeneration.The ideal leaf ages was 10 to 30 days of ’Toyonoka’ and ’Guinugan’ leaf.The ideal leaf ages was 10 to 20 days of ’Xuemi’ leaf.The ideal leaf ages was 10 to 20 days of the three varieties petiole.There was no effect of leaf burl on adventitious shoot regeneration of strawberry.2.The region of rd29A gene promoter was amplified by PCR technique.Sequence analysis showed that the fragment contained the main regulation motifs.Two a-ntisense expression vectors called pYH4215-rd29A and pYF7713-Rdre were obtained.3.By optimizing the transformation system of strawberry,a high-efficient transformation system was established.The bacterial concentration of OD600=0.4 and 10 minutes was the best condition.The available concentration of Cb was 200mg/L to eliminate Agrobacterium tumefaciens.The corresponding concentration of Kan was 20mg/L to screen.Co-culture for 3 days.The optimum Co-culture medium was MS+TDZ(2.0mg/L)+IBA(0.2mg/L),the optimum medium for elongation was MS+6-BA(0.5mg/L)+NAA(0.1mg/L)+Kan(20mg/L)+Cb(200mg/L),the optimum root inducing medium was MS+IBA(0.1mg/L)+Kan(20mg/L)+Cb(200mg/L).4.The transgenic plants contained RdreB1BI were obtained.The result of PCR and RT-PCR showed that the target gene was successfully integrated into the genome of strawberry.The concentration of praline indicated cold resistance of transformation strawberry had improved.更多还原