抗虫基因植物表达载体构建、遗传转化及表达研究
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摘要
本研究在王彦平构建的植物表达载体基础上构建Bt抗虫基因表达载体,并转化烟草,对Bt基因表达水平进行检测。试验进一步以普通741杨和转双抗虫基因pB29无菌苗为试验材料,建立了叶片再生体系并对农杆菌介导的遗传转化体系进行了优化。通过农杆菌介导法将Bt cry3A (Bt3)基因转入已含Bt cry1A(c) (Bt1)基因的pB29中,获得转双Bt基因741毛白杨,对部分转化植株进行了DNA水平和蛋白质水平的检测。主要研究结果如下:
利用植物表达载体pCAMBIA1305-Bt1-Bt3、pCAMBIA1305-Bt1和pCAMBIA1305-Bt3,通过DNA重组技术,分别将Bt1和Bt3基因正向插入植物表达载体pCAMBIA3301中,成功构建了pCAMBIA3301-Bt1、pCAMBIA3301-Bt3植物表达载体。此可读框架通过组成型启动子CaMV35S启动,携带PPT乙酰基转移酶(PAT)基因作为选择标记基因,并将构建的表达载体导入根癌农杆菌EHA105。
确定烟草转化的潮霉素临界筛选浓度为50 mg.L-l,头孢噻肟钠的抑菌浓度为200 mg.L-l。采用农杆菌介导法,将植物表达载体载体pCAMBIA1305-Bt1-Bt3、pCAMBIA1305-Bt1和pCAMBIA1305-Bt3及构建的植物表达载体pCAMBIA3301-Bt1、pCAMBIA3301-Bt3转入烟草中,均得到完整再生植株。经PCR检测,初步证明目的基因已整合到烟草的基因组中。ELISA检测表明,大部分转基因株系中Bt1和Bt3毒蛋白表达量均高于对照。在5株携带双价Bt基因的转基因烟草中,Bt1毒蛋白和Bt3毒蛋白的表达量最高分别达0.030 1%和0.293 8%。
对农杆菌介导的741杨遗传转化体系进行了优化,确定叶片诱导分化不定芽的适宜培养基为MS+ 6-BA 1.0 mg.L-l + NAA 0.1 mg.L-l,生根培养基为1/2 MS+ NAA 0.1 mg.L-l。潮霉素临界筛选浓度为10 mg.L-l,抗生素头孢噻肟钠的抑菌浓度为400 mg.L-l。遗传转化的适宜菌液浓度为OD600 = 0.4,浸菌时间为8~10 min,共培养时间以2 d为宜。
采用农杆菌介导法,将Bt3基因转入已转Bt1基因的杂种741毛白杨无性系pB29中,获得转双Bt基因741毛白杨。在含Hyg的培养基中进行多次继代筛选,获得抗性稳定的无性系9个,编号为pC1~pC9。
采用特异引物分别对获得的转双Bt基因741杨无性系进行PCR检测,结果表明:Bt1基因稳定存在于pB29无性系中,Bt3基因已整合到各无性系的基因组DNA中。对杂种741毛白杨转化植株进行毒蛋白表达的ELISA检测,结果表明,所选转双价Bt基因的741杨的2个无性系pC1和pC2,均检测到Bt1和Bt3毒蛋白的表达。Bt1毒蛋白的表达量分别为0.009 4%和0.011 0%;Bt3毒蛋白的表达量分别为0.217 0%和0.149 4%,高于对照pB29及转单一Bt3基因的741杨无性系CC71的Bt3蛋白表达量(0.033 7%)。
On the base of the plant expression vectors constructed by Wang Y P, the plant expression vectors harboring Bt genes were constructed and transformed into tobacco using leaf-disk method. The Bt genes expression levels were tested. Selecting 741 poplar and pB29 clone as the experiment materials, the leaf regeneration systems were established and optimized the Agrobacterium tumefaciens mediated transformation system. Mediated by Agrobacterium tumefaciens, Bt cry3A (Bt3) gene was integrated into pB29 genome which have harbored Bt cry1A(c) (Bt1) gene already and transgenic 741 poplars with two Bt genes were obtained. Some transgenic plants were tested on DNA and protein levels. The main results were as follows:
On the base of the plant expression vectors pCAMBIA1305-Bt1-Bt3, pCAMBIA1305-Bt1 and pCAMBIA1305-Bt3, using DNA recombination technology, the plant expression vector pCAMBIA3301-Bt1 fused Bt1 gene and the plant expression vector pCAMBIA3301-Bt3 fused Bt3 gene were separaterly constructed. Each gene was promoted with CaMV35S promoter, containing PAT gene as a selective marker. The plant expression vectors were transformed into EHA105.
Hygromycin (Hyg) critical concentration was 50 mg.L-l and Cefotaxime (CTX) was 200 mg.L-l for the tobacco transformation. Mediated by Agrobacterium tumefaciens, using the plant expression vectors pCAMBIA1305-Bt1-Bt3, pCAMBIA1305-Bt1 and pCAMBIA1305-Bt3 and the plant expression vectors pCAMBIA3301-Bt1 and pCAMBIA3301-Bt3, the Bt genes were transformed into tobacco using leaf-disk method and the Hyg and PPT resistant plants have been regenerated. PCR detections indicated that Bt1 and Bt3 genes were integrated into the tobacco genome. ELISA analysis showed that in most of the transgenic tobacco plants the expression levels of Bt proteins were higher than the comparison. In five transformed Bt1-Bt3 genes tobacco plants, both Bt1 and Bt3 proteins expressed in three plants. The expression levels of Bt1 and Bt3 proteins were higher than that in the wild-type tobacco plant. The highest could be up to 0.030 1% and 0.293 8% of the leaf total soluble proteins, respectively.
The leaf regeneration system of the 741 poplar was established and the Agrobacterium tumefaciens mediated transformation system was optimized. The shoot induction medium was MS+ 6-BA 1.0 mg.L-l + NAA 0.1 mg.L-l and the root induction medium was 1/2 MS+NAA 0.1 mg.L-l. Hygromycin (Hyg) critical concentration was 10 mg.L-l and Cefotaxime (CTX) 400 mg.L-l was a suitable concentration to control the propagation of Agrobacteria. Several crucial factors influencing the transformation efficiency were studied. It was found that OD600=0.4, infection of leaf explants for 8~10 min with Agrobacteria and cocultivation for 2 days after infection would befavorable for the transformation.
Mediated by Agrobacterium tumefaciens, Bt3 gene was integrated into pB29 genome, which had already integrated Bt1 gene already, then transgenic 741 poplars with two Bt genes were obtained. Through several transformations and selections, 9 clones of transgenic 741 poplar with two Bt genes were obtained and the clone numbers were pC1~pC9.
The results of PCR detection using special primers confirmed that Bt1 gene stably consisted in pB29 clones and Bt3 gene were integrated into the pB29 genome. ELISA analysis indicated that in the two clones pC1and pC2, both Bt1 and Bt3 proteins expressed. The expression levels of Bt1 protein were 0.009 4% and 0.011 0%, respectively. The expression levels of Bt3 proteins were 0.217 0% and 0.149 4%, higher than that of pB29 and that of the transformed single Bt3 gene 741 poplar clone CC71, of which the expression level of Bt3 protein was 0.033 7%.
利用植物表达载体pCAMBIA1305-Bt1-Bt3、pCAMBIA1305-Bt1和pCAMBIA1305-Bt3,通过DNA重组技术,分别将Bt1和Bt3基因正向插入植物表达载体pCAMBIA3301中,成功构建了pCAMBIA3301-Bt1、pCAMBIA3301-Bt3植物表达载体。此可读框架通过组成型启动子CaMV35S启动,携带PPT乙酰基转移酶(PAT)基因作为选择标记基因,并将构建的表达载体导入根癌农杆菌EHA105。
确定烟草转化的潮霉素临界筛选浓度为50 mg.L-l,头孢噻肟钠的抑菌浓度为200 mg.L-l。采用农杆菌介导法,将植物表达载体载体pCAMBIA1305-Bt1-Bt3、pCAMBIA1305-Bt1和pCAMBIA1305-Bt3及构建的植物表达载体pCAMBIA3301-Bt1、pCAMBIA3301-Bt3转入烟草中,均得到完整再生植株。经PCR检测,初步证明目的基因已整合到烟草的基因组中。ELISA检测表明,大部分转基因株系中Bt1和Bt3毒蛋白表达量均高于对照。在5株携带双价Bt基因的转基因烟草中,Bt1毒蛋白和Bt3毒蛋白的表达量最高分别达0.030 1%和0.293 8%。
对农杆菌介导的741杨遗传转化体系进行了优化,确定叶片诱导分化不定芽的适宜培养基为MS+ 6-BA 1.0 mg.L-l + NAA 0.1 mg.L-l,生根培养基为1/2 MS+ NAA 0.1 mg.L-l。潮霉素临界筛选浓度为10 mg.L-l,抗生素头孢噻肟钠的抑菌浓度为400 mg.L-l。遗传转化的适宜菌液浓度为OD600 = 0.4,浸菌时间为8~10 min,共培养时间以2 d为宜。
采用农杆菌介导法,将Bt3基因转入已转Bt1基因的杂种741毛白杨无性系pB29中,获得转双Bt基因741毛白杨。在含Hyg的培养基中进行多次继代筛选,获得抗性稳定的无性系9个,编号为pC1~pC9。
采用特异引物分别对获得的转双Bt基因741杨无性系进行PCR检测,结果表明:Bt1基因稳定存在于pB29无性系中,Bt3基因已整合到各无性系的基因组DNA中。对杂种741毛白杨转化植株进行毒蛋白表达的ELISA检测,结果表明,所选转双价Bt基因的741杨的2个无性系pC1和pC2,均检测到Bt1和Bt3毒蛋白的表达。Bt1毒蛋白的表达量分别为0.009 4%和0.011 0%;Bt3毒蛋白的表达量分别为0.217 0%和0.149 4%,高于对照pB29及转单一Bt3基因的741杨无性系CC71的Bt3蛋白表达量(0.033 7%)。
On the base of the plant expression vectors constructed by Wang Y P, the plant expression vectors harboring Bt genes were constructed and transformed into tobacco using leaf-disk method. The Bt genes expression levels were tested. Selecting 741 poplar and pB29 clone as the experiment materials, the leaf regeneration systems were established and optimized the Agrobacterium tumefaciens mediated transformation system. Mediated by Agrobacterium tumefaciens, Bt cry3A (Bt3) gene was integrated into pB29 genome which have harbored Bt cry1A(c) (Bt1) gene already and transgenic 741 poplars with two Bt genes were obtained. Some transgenic plants were tested on DNA and protein levels. The main results were as follows:
On the base of the plant expression vectors pCAMBIA1305-Bt1-Bt3, pCAMBIA1305-Bt1 and pCAMBIA1305-Bt3, using DNA recombination technology, the plant expression vector pCAMBIA3301-Bt1 fused Bt1 gene and the plant expression vector pCAMBIA3301-Bt3 fused Bt3 gene were separaterly constructed. Each gene was promoted with CaMV35S promoter, containing PAT gene as a selective marker. The plant expression vectors were transformed into EHA105.
Hygromycin (Hyg) critical concentration was 50 mg.L-l and Cefotaxime (CTX) was 200 mg.L-l for the tobacco transformation. Mediated by Agrobacterium tumefaciens, using the plant expression vectors pCAMBIA1305-Bt1-Bt3, pCAMBIA1305-Bt1 and pCAMBIA1305-Bt3 and the plant expression vectors pCAMBIA3301-Bt1 and pCAMBIA3301-Bt3, the Bt genes were transformed into tobacco using leaf-disk method and the Hyg and PPT resistant plants have been regenerated. PCR detections indicated that Bt1 and Bt3 genes were integrated into the tobacco genome. ELISA analysis showed that in most of the transgenic tobacco plants the expression levels of Bt proteins were higher than the comparison. In five transformed Bt1-Bt3 genes tobacco plants, both Bt1 and Bt3 proteins expressed in three plants. The expression levels of Bt1 and Bt3 proteins were higher than that in the wild-type tobacco plant. The highest could be up to 0.030 1% and 0.293 8% of the leaf total soluble proteins, respectively.
The leaf regeneration system of the 741 poplar was established and the Agrobacterium tumefaciens mediated transformation system was optimized. The shoot induction medium was MS+ 6-BA 1.0 mg.L-l + NAA 0.1 mg.L-l and the root induction medium was 1/2 MS+NAA 0.1 mg.L-l. Hygromycin (Hyg) critical concentration was 10 mg.L-l and Cefotaxime (CTX) 400 mg.L-l was a suitable concentration to control the propagation of Agrobacteria. Several crucial factors influencing the transformation efficiency were studied. It was found that OD600=0.4, infection of leaf explants for 8~10 min with Agrobacteria and cocultivation for 2 days after infection would befavorable for the transformation.
Mediated by Agrobacterium tumefaciens, Bt3 gene was integrated into pB29 genome, which had already integrated Bt1 gene already, then transgenic 741 poplars with two Bt genes were obtained. Through several transformations and selections, 9 clones of transgenic 741 poplar with two Bt genes were obtained and the clone numbers were pC1~pC9.
The results of PCR detection using special primers confirmed that Bt1 gene stably consisted in pB29 clones and Bt3 gene were integrated into the pB29 genome. ELISA analysis indicated that in the two clones pC1and pC2, both Bt1 and Bt3 proteins expressed. The expression levels of Bt1 protein were 0.009 4% and 0.011 0%, respectively. The expression levels of Bt3 proteins were 0.217 0% and 0.149 4%, higher than that of pB29 and that of the transformed single Bt3 gene 741 poplar clone CC71, of which the expression level of Bt3 protein was 0.033 7%.
引文
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