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双抗虫基因转化欧美杨107杨的研究

标题: 双抗虫基因转化欧美杨107杨的研究
英文标题: The Research on Two Insect-resistant Gene Transforming into Populus*euramericana Cv."74/76"
作者: 杨艳丽
出版时间: 2012-01-01
所在大学: 河北农业大学
关键词: 欧美杨107杨,农杆菌,遗传转化,再生体系,检测
英文关键词: Poplar 107,Agrobacterium tumefaciens,Genetic transformation,Regeneration system,detection
论文级别: 硕士
学位: 学位论文
导师: 刘桂林%梁海永
专业: 园林植物与观赏园艺
提交时间: 2012
摘要: 杨树在全球分布十分广泛,是重要的造林树种和生态防护林树种.其树干通直,树形优美,是很好的园林绿化树种,广泛应用于园林中,通常做植物造景树和景观背景树.杨树由于其速生性以及高度的观赏价值在园林绿化和经济林中得到了越来越广泛的应用,然而,近年来,病虫害的侵犯也造成了重大的损失.转双抗虫基因欧美杨107杨的获得不但能有效地解决杨树的虫害问题,而且该树种的速生性也将使其成为园林绿化应用的首选树种,转双抗虫基因欧美杨107杨的获得不管是从社会需求还是从经济效益上考虑,它的研发和培育都是必要的.目前,在杨树的众多树种中,只有白杨派树种在组织培养和遗传转化方面相对成熟.本试验主要采用农杆介导法将构建在一个载体上的双抗虫基因(BtCry1Ac基因+API慈姑蛋白酶抑制剂基因)对欧美杨107杨进行遗传转化研究,并对转基因植株进行了分子生物学检测和初步饲虫试验,获得了转双抗虫基因的欧美杨107杨株系.(1)针对影响欧美杨107杨的遗传转化的不同因素,初步建立了欧美杨107杨品种高效组织培养再生体系及遗传转化体系,以材料的茎段和叶片为外植体,通过筛选获得最佳分化培养基,建立了其稳定高效的再生体系,结果表明:不定芽诱导分化最佳培养基为MS+6-BA 0.5mg/L+IAA 0.15mg/L+蔗糖25g/L+琼脂7g/L,确定生根培养基为1/2MS+IBA0.5mg/L+蔗糖15g+琼脂7g/L.同时研究结果表明:叶片主叶脉及叶柄和茎段伤口处发芽状况相似,因此选取叶片或茎段作为试验材料对本次试验影响不大.(2)采用农杆菌介导法进行基因转化,对影响转化的各个因素进行了优化研究.结果表明:在浸菌15min,共培养3d,得到抗性芽,诱导率为16.7%.经过卡那霉素的筛选,生根培养,炼苗和移栽,获得抗性植株.(3)根据PCR分析及ELISA毒蛋白检测结果,证明Bt基因和蛋白酶抑制剂基因已经整合到欧美杨107杨基因组DNA中.通过本试验的研究,获得了转双抗虫基因的欧美杨107杨10个系号,其中10个系号进行PCR检测都为阳性, ELISA检测10个系号中毒蛋白含量测定占总蛋白最高表达量为0.0336%.(4)用转基因欧美杨107杨的幼嫩叶片喂养美国白蛾1龄幼虫的虫试结果表明,转双抗虫基因欧美杨107杨对幼虫的毒杀率明显高于对照未转基因杨树,6个编号的植株叶片被美国白蛾取食后,其中4个号系死亡率能达到100%.说明这2个基因在植物细胞内均能够有效地表达,其表达产物对美国白蛾幼虫具有较强的毒性.(5)经分子生物学检测和虫试效果,证明了目的基因已经整合到欧美杨107杨的基因组中,从转基因株系中筛选出生长发育正常并抗虫能力强的优良株系.
英文摘要: Poplars is widely distributed in the global and important afforestation and ecological shelterbelt species. Due to its straight trunk and beautiful tree shape Poplar is a good landscape trees and widely used in the garden, which usually be took as plant landscaping trees and landscape background tree. Because of its feature of rapid growth and high ornamental value, Poplar has been more widely used in landscaping and economic forest. However, in recent years, the violations of pests and diseases have caused a significant loss. The obtain of transgenic hybrid poplar 107 pest can not only effectively solve the issue of pest but also take poplar as preferred specie in landscaping application due to its rapid growth nature. From the respect of social needs or economic benefits, the culture and research of transgenic hybrid poplar 107 were necessary.At present, in many species of poplar, only Leuce Duby species is relatively mature in tissue culture and genetic transformation. This exeperiment mainly used agricultural rodmediated method to do Genetic transformation research of Europe and the United States Yang 107 Populus by using bi-insect-resistant genes built in a vector (BtCry1Ac gene +API arrowhead protease inhibitor gene), for transgenic plants, molecular biology detect and preliminary feeding insects trial had been took, and had obtained transgenic hybrid poplar 107 lines.(1) According to different factors influencing genetic transformation of Europe and America 107 poplar, efficient tissue culture regeneration and genetic transformation system of 107 Populus varieties were initially established. By means of taking Material stems and leaves as explants, through selecting the best medium of their various growth stages, stable and efficient regeneration system was established. The results show that: optimal medium for adventitious buds induced differentiation was MS+6-BA 0.5 mg/L+IAA 0.15 mg/L+sucrose 25 g/L+ agar 7 g/L, for rooting medium was 1/2 MS+IBA 0.5 mg/L+ sucrose 15 g+ agar 7 g/L. Simultaneously the results showed that: main veins, germination situation in leaf petioles and stems wound were similar. Therefore, selecting a leaf or stem segments as the test materials had little effect on the experiment.(2) Agrobacterium-mediated method was used for genetic transformation, Optimization research of the various factors affecting transformation had been took. The results show that: were being baptist bacteria 15min and culturing 3 dayas altogether, resistant shoots were obtained with induction rate 16.7%. After kanamycin screening, rooting culture, hardening and transplanting, obtain resistant plants were acquiring.(3) According to PCR analysis and ELISA experimental results, the Bt gene and protease inhibitor genes had been integrated into Europe and America 107 poplar genomic DNA was proved. Through this research, we obtained transgenic hybrid Europe and America 107 poplar 10 clones. Among these7 clones was positive in the PCR assay. In ELISA detection of 10 departments, the highest expression levels of toxic protein content occupying in the total protein was 0.0336%.(4) Insects test of feeding the Hyphantria instar larvae on leaves of Europe and America 107 poplar show that deratization rate of Transgenic hybrid Europe and America 107 poplar was significantly higher than the control not genetically modified. 6 numbered leaves being feed fall webworm, among these 4 numbers mortality can reach 100%. Above of these show it is a fact that these two genes in plant cells were able to effectively express and its expression products had a strong toxicity for the fall webworm larvae.(5) Through molecular biology detection and insect test results, the target gene had been integrated into the genome of Europe and America 107 poplar was proved, and having normal growth and development and the ability of insect-resistant fine strains had been screened out from the transgenic lines .