抗草甘膦基因甜菜遗传转化的研究

【作者】 芦宝琳；

【导师】 李北齐； 程大友；

【作者基本信息】 黑龙江大学 ， 植物保护， 2012， 硕士

【摘要】 种植田中的杂草是导致甜菜产量和含糖量下降的重要原因之一。通过基因工程手段获得抗草甘膦除草剂的甜菜品种成为有效的除去田间杂草，解决杂草对甜菜产量和含糖的影响。本研究以甜菜无菌苗的叶柄为外植体材料，通过农杆菌介导方法将EPSPS基因转入甜菜外植体内，对农杆菌转化条件、方法及甜菜外植体再生条件进行较为系统地比较研究，建立与优化了转基因甜菜再生体系，从而获得转基因甜菜的再生植株。1、转基因甜菜再生体系的建立与优化本研究利用DM1-9、O-68和DY5-O三种不同基因型的甜菜品系为试验材料。通过对三个品系甜菜不同部位、不同培养基和不同苗龄进行试验，总结出最适合应用于转基因条件的甜菜品系为DM1-9和O-68。转基因甜菜材料外植体制备用无菌苗培养的优化方法为：以DM1-9和O-68为材料，利用筛选出的再生培养基MS+1mg/L6-BA+0.1mg/L NAA+100mg/LKan+500mg/LCb、继代培养基MS+6-BA0.5mg/L+NAA1.0mg/L+100mg/LKan+500mg/LCb及生根培养基MS+1.0mg/LNAA使得该甜菜品系达到最大的转化效率。通过农杆菌侵染转基因试验，筛选出转基因操作时最佳的农杆菌侵染条件为：OD600=0.3时侵染10min。2、外植体中农杆菌的最佳去除方法筛选出农杆菌与甜菜外植体共培养操作后的农杆菌最佳去除方法为：500mg/LCB清洗1次无菌水冲洗3次，300mg/LCef清洗1遍，无菌水冲洗3次后用无菌滤纸吸干。3、获得转入EPSPS基因的甜菜再生植株利用农杆菌侵染方法，根据建立的最佳遗传转化方法和条件进行甜菜转基因试验，获得转抗草甘膦基因的甜菜植株。对所获得的卡那霉素抗性转基因再生植株中提取的总DNA进行PCR检测，以EPSPS基因两端序列设计引物，部分卡那霉素抗性再生植株扩增出目的基因片段。更多还原

【Abstract】 Weed in planting fields is one of the most important reasons for the sugar beetyield and sugar content decrease. Getting glyphosate herbicide resistance sugar beetvarieties by means of genetic engineering method is one of the effective solutions todeal with this problem. Sugar beet seeding petioles were used as transgene explantmaterials in this study. EPSPS gene was delivered into sugar beet explants byagrobacterium mediated method. Agrobacterium culture transform method and shootsregeneration conditions from beet explants were systematically studied in this paper.Transgenic beet regeneration system was established and optimized. transgenic sugarbeet plants were obtained.1、Establishment and optimization of sugar beet shoots regeneration systemThree different genotypes sugar beet lines (DM1-9, O-68and DY5-O) were used astransgene experiment materials in this study. The result showed that the most suitabletransgenic sugar beet lines were DM1-9and O-68according to experiments of sugarbeet different expants, different media and different seedling age. DM1-9and O-68were used as experiment materals in following experiment. Sugar beet shootsregeneration system was established list as follows. Shoots regeneration medium wasMS+1.0mg/L6-BA+0.1mg/L NAA+100mg/LKan+500mg/LCb. Subculturemedium was MS+6-BA0.5mg/L+NAA1.0mg/L+100mg/LKan+500mg/L Cb.Rooting culture medium was MS+1.0mg/L NAA. The maximum conversionefficiency was obtained by above system. The result of agrobacterium infectionexperment showed that transgenic operation optimal Agrobacterium infectionconcentration was OD600=0.3and infection time was10minute.2、The best method of agrobacterium removal from explantsThe agrobacterium-mediated trangene experiment result showed that the optimal removal method of agrobacterium removal after beet explant co-culture operation waslisted as follows:500mg/L Cb solution cleaning the explant, then sterile water rinse3times,300mg/L Cef solution washing1times, sterile water rinse explants treetimes. Water was suck dry with sterile filter paper at last.3、Getting transgene regeneration sugar beet plantsUsing Agrobacterium infection technology, sugar beet transgene experiment wasconducted in line with above optimization genetic transformation motheds. Glyphosateresistant sugarbeet planst were obtained. PCR detection was conducted with EPSPSgene among kanamycin resistance transgenic regenerated shoots plants. EPSPSfragment was amplified in some regeneration plants by PCR detection.更多还原