基于抗草甘膦基因的棉花茎尖农杆菌遗传转化方法的研究
发布时间：2019-05-11 06:22
【摘要】：棉花是一种重要的经济作物,在我国国民经济发展等方面有着至关重要的作用。草害是影响棉花生产的主要因素之一,对棉花的产量及品质有较大的影响。运用基因工程创新棉花种质资源,培育应用转基因抗除草剂的棉花品种对于棉田化学除草,降低植棉投入,提高植棉效率具有重要的意义。本研究以具有自主知识产权的抗草甘膦除草剂基因(EPSPS-G6)为外源基因,通过优化多种转化条件,完善了基于抗草甘膦基因的棉花茎尖农杆菌遗传转化技术,并将EPSPS-G6基因导入珂字棉201、YZ-1及中棉所49等棉花品种中,创制出抗草甘膦转基因棉花种质系。研究结果如下： 1.以中棉所49、珂字棉201及YZ-1为受体材料,通过对培养基草甘膦浓度的筛选、农杆菌侵染浓度、共培养时间以及恢复培养条件等因素的优化,完善了基于抗草甘膦基因的棉花茎尖农杆菌遗传转化技术。研究结果表明,适合于棉花茎尖农杆菌遗传转化法的草甘膦筛选浓度为10mg/L；茎尖转化体系：以20min为农杆菌侵染时间,农杆菌菌液OD600为0.9-1.0,共培养时间为48h,选用SH培养基并加入适量活性炭(0.5g/L)作为恢复培养基。以茎尖外植体为基数,本转化技术的转化成功率达6.4%。从转化时间来看,该方法从无菌苗培养开始计算,获得再生试管苗的时间仅为2个月时间,4个半月即能开花,6个月即能获得T1代种子,说明本转化技术具有较好的实用价值。 2.以本研究建立的棉花茎尖农杆菌遗传转化技术,将具有我校自主知识产权的抗草甘膦基因EPSPs-G6导入中棉所49、珂字棉201及YZ-1等3个陆地棉品种(系)的茎尖。共转化360个茎尖,共获得60株抗性再生植株,经分子检测和草甘膦筛选后移栽成活23株。 3.对再生植株(T0)的基因组DNA进行PCR扩增,26株再生植株中能扩增出与质粒DNA相同大小条带,初步说明外源基因已成功地整合到再生植株的染色体DNA中。Southern杂交鉴定结果表明,转基因植株的外源基因均为单拷贝。转基因T0植株主要田间农艺性状调查结果表明,转EPSPS-G6基因T0当代生长正常,全部能开花结实,并获得T1种子。此外,23株转基因植株整体性状与受体对照无明显差异,不同转化体之间无明显的变异。说明本研究创造的抗草甘膦棉花种质具有较好的育种利用价值。
[Abstract]:Cotton is an important cash crop, which plays an important role in the development of our national economy. Grass damage is one of the main factors affecting cotton production, and has a great impact on cotton yield and quality. Using genetic engineering to innovate cotton germplasm resources and cultivate cotton varieties with transgenic herbicide resistance are of great significance for chemical weeding in cotton fields, reduction of cotton planting input and improvement of cotton planting efficiency. In this study, glyphosate resistant herbicide gene (EPSPS-G6) with independent intellectual property rights was used as foreign gene, and the genetic transformation technology of Agrobacterium tumefaciens based on glyphosate resistance gene was improved by optimizing various transformation conditions. The transgenic cotton germplasm lines resistant to glyphosate were established by introducing EPSPS-G6 gene into cotton varieties such as Ke cotton 201,YZ 1 and Zhongmiansuo 49. The results showed that the transgenic cotton germplasm was resistant to glyphosate. The results are as follows: 1. The glyphosate concentration, Agrobacterium tumefaciens infection concentration, co-culture time and recovery culture conditions were optimized by screening glyphosate concentration, co-culture time and recovery culture conditions. The genetic transformation technology of Agrobacterium tumefaciens based on glyphosate resistance gene was improved. The results showed that the screening concentration of glyphosate suitable for Agrobacterium tumefaciens genetic transformation was 10 mg 路L ~ (- 1). Stem tip transformation system: using 20min as Agrobacterium tumefaciens infection time, Agrobacterium tumefaciens solution OD600 0.9 鈮，
本文编号：2474313
[Abstract]:Cotton is an important cash crop, which plays an important role in the development of our national economy. Grass damage is one of the main factors affecting cotton production, and has a great impact on cotton yield and quality. Using genetic engineering to innovate cotton germplasm resources and cultivate cotton varieties with transgenic herbicide resistance are of great significance for chemical weeding in cotton fields, reduction of cotton planting input and improvement of cotton planting efficiency. In this study, glyphosate resistant herbicide gene (EPSPS-G6) with independent intellectual property rights was used as foreign gene, and the genetic transformation technology of Agrobacterium tumefaciens based on glyphosate resistance gene was improved by optimizing various transformation conditions. The transgenic cotton germplasm lines resistant to glyphosate were established by introducing EPSPS-G6 gene into cotton varieties such as Ke cotton 201,YZ 1 and Zhongmiansuo 49. The results showed that the transgenic cotton germplasm was resistant to glyphosate. The results are as follows: 1. The glyphosate concentration, Agrobacterium tumefaciens infection concentration, co-culture time and recovery culture conditions were optimized by screening glyphosate concentration, co-culture time and recovery culture conditions. The genetic transformation technology of Agrobacterium tumefaciens based on glyphosate resistance gene was improved. The results showed that the screening concentration of glyphosate suitable for Agrobacterium tumefaciens genetic transformation was 10 mg 路L ~ (- 1). Stem tip transformation system: using 20min as Agrobacterium tumefaciens infection time, Agrobacterium tumefaciens solution OD600 0.9 鈮，
本文编号：2474313
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