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病害是造成烟草减产和品质降低的主要因素,而利用生物技术提高烟草抗病性是解决此问题的有效途径,其中包括利用与植物抗病性密切相关的关键因子如病程相关基因非表达子(non-expressor of pathogenesis-related genes1,NPR1)。本研究利用源于海岛棉的Gb NPR1基因,表皮特异性启动子CUT1和组成型启动子35S构建两个植物表达载体35S-Gb NPR1和CUT1-Gb NPR1,经农杆菌介导法分别转化烟草。T0代及T1代转化植株PCR检测证明,目的基因已整合到核基因组中。T1代植株RT-PCR分析表明,Gb NPR1基因在转录水平得以表达。bar试纸条检测为阳性,证明和Gb NPR1连锁的草甘膦基因能够正常转录和翻译表达。将阳性植株进行烟草病菌赤星病、炭疽病和低头黑病等三种病害的病原菌离体接菌实验,研究结果表明:转CUT1-Gb NPR1载体的转基因烟草虽然较转35S-Gb NPR1载体的烟草对三种病菌的抗性稍低,但较野生型烟草相比具有较高抗性。转Gb NPR1基因的烟草能提高对低头黑病和炭疽病的抗性为首次报道。
Disease is the main factor that causes the decrease of yield and quality of tobacco, and the use of biotechnology to improve the resistance of tobacco is an effective way to solve this problem, including the use of key factors closely related to plant disease resistance such as disease-related gene non-expression non-expressor of pathogenesis-related genes 1, NPR1). In this study, two plant expression vectors 35S-Gb NPR1 and CUT1-Gb NPR1 were constructed using Gb NPR1, CUT1, and 35S constitutive promoters derived from island cotton. The tobacco plants were transformed by Agrobacterium-mediated method. T0 generation and T1 generation of transgenic plants PCR test showed that the target gene has been integrated into the nuclear genome. RT-PCR analysis of T1 plants showed that Gb NPR1 gene was expressed at the transcriptional level. bar test strip was positive, demonstrating that the glyphosate gene linked to Gb NPR1 is normally transcribed and translated. The positive plants were inoculated with the pathogenic bacteria of three pathogenic diseases, brown spot disease, anthracnose and blackhead disease. The results showed that the transgenic tobacco transformed with CUT1-Gb NPR1 was more stable than the tobacco with 35S-Gb NPR1 vector The resistance to the three pathogens was slightly lower, but higher than the wild type tobacco. For the first time, tobacco that transferred Gb NPR1 gene to improve the resistance to DL and anthracnose was reported.