抗草甘膦转基因玉米的研究

发布时间：2018-04-11 08:20

本文选题：玉米 + CP4-EPSPS基因　；参考：《山西大学》2016年硕士论文

【摘要】：玉米(Zea mays L.)作为世界三大粮食作物之一,是重要的粮食作物、饲料以及工业加工原料。而杂草是玉米减产的主要危害之一,在玉米生长过程中会争夺水分,养分和阳光,并且引起病虫害的发生。因而研究如何有效经济的控制玉米田间杂草具有重要意义。随着生物技术的发展,利用基因工程的手段获得抗草甘膦作物新品种已成为最理想的手段。自1998年,抗草甘膦转基因玉米品种GA21获得批准商业化种植以来,各种抗草甘膦、抗虫、抗病等优良性状的转基因玉米品种相继推出,转基因玉米是世界上种植面积最广的转基因粮食作物。本研究中利用超声波辅助花粉介导转化法和超声波辅助农杆菌介导萌发种子转化法两种方法将携带目的基因——抗草甘膦基因CP4-EPSPS的载体转入到玉米自交系昌7-2中。其中pM3301UbiSpCP4载体选用了组成型强启动子——玉米泛素启动子pZmUbi-1启动表达,同时该载体携带了叶绿体信号肽(Signal Peptide),有助于目的基因成功定位到叶绿体内,使得CP4基因在玉米叶绿体中更好的表达。另外这两种新型的转基因方法是以花粉和萌发的种子作为受体,避开了组织培养再生植株的过程,且无基因型依赖性,为广大育种工作者提供了简便经济且易于操作的两种种质创新途径。另外玉米自交系昌7-2作为目前生产中广泛使用的优良自交系,农艺性状良好,获得转基因株系后能最快应用于生产。主要研究结果如下:将构建好的载体pM3301UbiSpCP4利用超声波辅助花粉介导的转基因方法对1552株(T0代)玉米进行遗传转化,成功获得了943粒结实种子。对T1代出苗的648个转化株涂抹2%浓度的草甘膦进行田间鉴定筛选,有29株长势健康,表现出抗草甘膦的性状,通过PCR对这些株系进行目的基因检测,其中有16个株系扩增出阳性条带,RT-PCR显示16个阳性株系中外源基因CP4-EPSPS正常转录。同时进行了PCR-Southern blot检测,进一步证明了外源基因CP4-EPSPS稳定遗传到了T1代。T1代的16个株系自交后收获种子(T2代),对T2代部分转基因株系涂抹2%浓度的草甘膦,筛选出了5个抗性良好的转基因株系Anti-3,11,12,23,29。经PCR、Southern、ELISA检测结合田间抗草甘膦筛选试验,最终获得了3个高抗草甘膦、单拷贝且抗除草剂蛋白表达量较高的转基因玉米株系Anti-3,23,29。本研究为抗除草剂转基因玉米的研究奠定了基础,并为育种工作提供有价值的新种质。
[Abstract]:Zea mays L.As one of the three major food crops in the world, it is an important food crop, fodder and raw material for industrial processing.Weed is one of the main harms of maize yield reduction. It will compete for water, nutrients and sunlight in the process of maize growth, and cause the occurrence of diseases and insect pests.Therefore, it is of great significance to study how to control weeds in maize field economically.With the development of biotechnology, it has become the most ideal method to obtain new varieties of glyphosate resistant crops by genetic engineering.Since 1998, when glyphosate resistant transgenic maize (GA21) was approved for commercial planting, a variety of transgenic maize varieties with excellent characters, such as glyphosate resistance, insect resistance and disease resistance, have been introduced one after another.Transgenic corn is the most widely grown genetically modified food crop in the world.In this study, ultrasonic assisted pollen mediated transformation and ultrasonic assisted Agrobacterium tumefaciens seed transformation were used to transfer the vector carrying the target gene, glyphosate resistant gene CP4-EPSPS, into maize inbred line Chang7-2.Among them, the pM3301UbiSpCP4 vector selected the component strong promoter-maize ubiquitin promoter pZmUbi-1 to initiate expression, and the vector carried the chloroplast signal peptide Signal Peptidea, which was helpful for the target gene to be successfully located in the chloroplast.CP4 gene is better expressed in maize chloroplast.In addition, the two new transgenic methods used pollen and germinated seeds as receptors, avoiding the process of tissue culture and regeneration, and without genotypic dependence.It provides a simple, economical and easy to operate ways for breeders to innovate germplasm.In addition, the maize inbred line Chang7-2 is widely used in production at present. It has good agronomic characters and can be used in production as soon as transgenic lines are obtained.The main results are as follows: 943 seed bearing seeds were successfully obtained by genetic transformation of 1552 maize plants in T0 generation by using the constructed vector pM3301UbiSpCP4.Field identification and screening of 648 transgenic plants with 2% concentration of glyphosate were carried out in T1 generation. 29 of them had healthy growth and showed resistance to glyphosate. The target genes of these lines were detected by PCR.16 of them amplified positive bands and RT-PCR showed normal transcription of foreign gene CP4-EPSPS in 16 positive lines.At the same time, PCR-Southern blot analysis showed that the exogenous gene CP4-EPSPS was stably inherited to 16 lines in T1 generation and T1 generation, and then harvested the seeds of T 2 generation, and applied 2% glyphosate to some transgenic lines in T2 generation.Five transgenic lines with good resistance were screened out.Three transgenic maize lines with high glyphosate resistance, single copy and high herbicide protein expression were obtained by PCR Southern Elisa combined with field glyphosate resistant screening test.This study laid a foundation for the study of herbicide resistant transgenic maize and provided valuable new germplasm for breeding.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S513

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