番茄抗根结线虫基因SlMi导入莴苣的研究

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植物根结线虫(Meloidogyne spp.)是一种专性寄生病害,可寄生于3000多种植物,是世界四大植物侵染性病原之一,给我国和世界农业带来了巨大的经济损失。本研究根据同源序列法从番茄中克隆到抗根结线虫基因SlMi,并建立了莴苣高效再生和遗传转化体系,将该基因通过农杆菌介导的遗传转化法转入到缺乏抗性资源的莴苣中,为莴苣的抗根结线虫育种奠定了基础,并对异源基因在莴苣中的成功应用提供了范例。主要研究结果如下:1.建立了莴苣的再生与转化体系。通过对16种培养基的筛选比较发现,不同的莴苣品种在不同的培养基中其分化频率有较大差别。其中耐热二白皮的分化能力最强,在7种培养基中的分化频率均超过了90%,其最优分化培养基为MS+6-BA0.5mg/L+NAA0.20 mg/L,分化频率达99.21%;华选一号次之,在4种培养基中的分化频率超过90%,其最优分化培养基为MS+6-BA 1.0 mg/L+NAA 0.25 mg/L,分化频率达97.58%;耐抽薹生菜在3种培养基中的分化频率超过90%,其最优分化培养基为MS+6-BA0.5 mg/L+NAA0.25 mg/L,分化频率达94.94%。以华选一号为受体进行了转化体系的建立,以Kam为100 mg/L时对抗性芽的筛选效果最好。2.根据GenBank中已公布的番茄根结线虫抗性基因Mi(AF0396821的序列信息,利用同源序列法克隆了该基因,并构建了SlMi基因的正义遗传转化载体。3.利用农杆菌介导的方法,成功的将番茄抗根结线虫基因SlMi导入到莴苣华选一号中,获得24棵卡那霉素抗性植株。对其中的4棵进行PCR和Southern Blot检测,结果表明在3棵植株中有外源基因的整合,其拷贝数1-3个不等。4.莴苣转基因植株的半定量RT-PCR分析结果表明,有3棵转化植株靶基因的表达量比对照明显增加。5.转基因植株的根结线虫抗性评价结果表明3棵转基因植株对根结线虫的抗性明显提高。6.对转基因植株T1代进行了种子发芽率、种子形态学的分析,结果表明单拷贝的株系L1、L4与对照基本没有差别,而多拷贝的株系L3则和对照有较大的差异。7.石蜡切片分析结果表明,根结线虫侵染后造成根部组织结构显著的变化。感病莴苣植株根部可以看到很多线虫,根结线虫周围有许多多核的巨细胞;而在转基因植株中则可看到因过敏性反应造成的坏死细胞。
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