农杆菌介导单、双价抗虫基因Bt、Bt-pinⅡ基因对观赏羽衣甘蓝的遗传转化研究
摘要
观赏羽衣甘蓝(Brassica oleracea var.acephala),属十字花科芸薹属甘蓝种的园艺变种,是一种赏食兼用的耐冻优良景观植物。羽衣甘蓝的在生长期易发生虫害,影响其生产及观赏价值。而目前的育种资源中,尚无相应的种质。要解决这两个问题,有希望的途径就是通过现代基因工程技术,引入外源抗性基因,创制新的抗性种质。但其育种研究尚处于起步阶段,国际上鲜有研究报道,缺乏针对育种目的、育种材料的详细研究资料。国内利用基因工程手段改良其性状还是一个空白。
本研究通过基因工程为技术手段,将所克隆的抗虫基因导入观赏羽衣甘蓝,赋予其新的性状,以期获得优良的抗虫种质资源。主要结论如下:
1.以8份不同类型羽衣甘蓝的优良育种品系为试材,建立高频再生体系,筛选出两份材料,其带柄子叶分别在L6(MS+6-BA 1.0 mg/L+NAA 0.1mg/L)及L3(MS+6-BA 1.5 mg/L)培养基中不定芽诱导率为100%,每外植体上的不定芽数高达25个。下胚轴诱导率最高也能达88.33%;
2.根据感染时间、共培养时间、外植体的脱菌和Basta选择压的确定等因素与转化频率的相关性研究,建立了适合于观赏羽衣甘蓝皱白1的根癌农杆菌介导的转化体系即:侵染时间3-5min;黑暗共培养2天;Basta筛选压为5mg/L;菌液OD值为0.3-0.5;
3.克隆了Bt基因和Bt-PinⅡ融合基因,构建了pBAC171、pBAC172和pBAC173三个植物表达载体。通过农杆菌介导法将基因分别导入羽衣甘蓝皱白1,分别获得7株单价和5株双价抗虫转基因植株;PCR扩增外源Bt和Bt-PinⅡ基因片段,结果都有目的条带;对7株含Bt基因的植株进行southern检测,有5株再生植株的外源基因片段整合到基因组中,其中有1～3个转基因拷贝。
Ornamental kale(Brassica oleracea var.acephala)is an edible freezing tolerant excellent landscape plant,belonging to cabbge's horticulture varieties of Cruciferae Brassica.It is so prone to occur pest damage and freezing injury in its growing season that its production and ornamental value will be influenced.At present,there is no corresponding breeding resources.To endow it with pest insect resistance and increase the minus temperature tolerance further,there are a way will be operated through modern genetic engineering technology to create new resistant germplasm. However,breeding of ornamental kale is still in its infancy,and few international studies have been reported,lacking of detailed research data.Domestic improving their characters is still vacancy using genetic engineering technology.
In this study,the plant expression vectors harboring insecticidal protein gene which were cloned were constructed and introduced into ornamental kale through genetic engineering technology,giving it new traits,to obtain good insect-resistant germplasm.The main results are as follows:
1.A high efficient shoot regeneration system was established with different explants of eight genotypes representing the main phenotypes of ornamental kale.Two genotypes with 100%shoot induction rate on L6 or L3 medium respectively using cotyledon with petiole as explants were screened out,and the no.of shoots per explant were up to 25.The feasible rooting medium was MS+NAA 0.1 mg/L and the rooting rate reached to 100%.
2.According to the studies on the relation between the infection time,the cultivation time,the sterilization of the explants,the ascertainment of ornamental kale the Basta screening pressure,the transgenic system of with Agrobacterium tumefaciens was constructed:infecting 3-5min;2d co-cultivation in dark;0.3-0.5 OD of bacterium..
3.Three plant expression vectors pBAC171 and pBAC172、pBAC173,harboring a Bt
本研究通过基因工程为技术手段,将所克隆的抗虫基因导入观赏羽衣甘蓝,赋予其新的性状,以期获得优良的抗虫种质资源。主要结论如下:
1.以8份不同类型羽衣甘蓝的优良育种品系为试材,建立高频再生体系,筛选出两份材料,其带柄子叶分别在L6(MS+6-BA 1.0 mg/L+NAA 0.1mg/L)及L3(MS+6-BA 1.5 mg/L)培养基中不定芽诱导率为100%,每外植体上的不定芽数高达25个。下胚轴诱导率最高也能达88.33%;
2.根据感染时间、共培养时间、外植体的脱菌和Basta选择压的确定等因素与转化频率的相关性研究,建立了适合于观赏羽衣甘蓝皱白1的根癌农杆菌介导的转化体系即:侵染时间3-5min;黑暗共培养2天;Basta筛选压为5mg/L;菌液OD值为0.3-0.5;
3.克隆了Bt基因和Bt-PinⅡ融合基因,构建了pBAC171、pBAC172和pBAC173三个植物表达载体。通过农杆菌介导法将基因分别导入羽衣甘蓝皱白1,分别获得7株单价和5株双价抗虫转基因植株;PCR扩增外源Bt和Bt-PinⅡ基因片段,结果都有目的条带;对7株含Bt基因的植株进行southern检测,有5株再生植株的外源基因片段整合到基因组中,其中有1～3个转基因拷贝。
Ornamental kale(Brassica oleracea var.acephala)is an edible freezing tolerant excellent landscape plant,belonging to cabbge's horticulture varieties of Cruciferae Brassica.It is so prone to occur pest damage and freezing injury in its growing season that its production and ornamental value will be influenced.At present,there is no corresponding breeding resources.To endow it with pest insect resistance and increase the minus temperature tolerance further,there are a way will be operated through modern genetic engineering technology to create new resistant germplasm. However,breeding of ornamental kale is still in its infancy,and few international studies have been reported,lacking of detailed research data.Domestic improving their characters is still vacancy using genetic engineering technology.
In this study,the plant expression vectors harboring insecticidal protein gene which were cloned were constructed and introduced into ornamental kale through genetic engineering technology,giving it new traits,to obtain good insect-resistant germplasm.The main results are as follows:
1.A high efficient shoot regeneration system was established with different explants of eight genotypes representing the main phenotypes of ornamental kale.Two genotypes with 100%shoot induction rate on L6 or L3 medium respectively using cotyledon with petiole as explants were screened out,and the no.of shoots per explant were up to 25.The feasible rooting medium was MS+NAA 0.1 mg/L and the rooting rate reached to 100%.
2.According to the studies on the relation between the infection time,the cultivation time,the sterilization of the explants,the ascertainment of ornamental kale the Basta screening pressure,the transgenic system of with Agrobacterium tumefaciens was constructed:infecting 3-5min;2d co-cultivation in dark;0.3-0.5 OD of bacterium..
3.Three plant expression vectors pBAC171 and pBAC172、pBAC173,harboring a Bt
引文
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