【作者】 李志兰；

【导师】 杨敏生；

【作者基本信息】 河北农业大学 ， 森林培育， 2003， 硕士

【摘要】 三倍体毛白杨是近年来我国大力推广的速生树种之一。一直以来三倍体毛白杨的抗虫性没有得到明显改善，随着栽培面积的不断扩大，多种食叶害虫的危害越来越严重。利用转基因技术培育抗虫杨树新品种已成为当前植物遗传改良的重要手段。本研究主要用构建在一个载体上的双抗虫基因（BtCryI基因和慈姑蛋白酶抑制剂基因API）对三倍体毛白杨进行了遗传转化，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三倍体毛白杨株系。 1 通过实验得到三倍体毛白杨的最佳再生体系是：以MS+6-BA0.6mg·^-1+NAA0.1 mg·L^-1附加1.5％蔗糖、0.6％琼脂作为三倍体毛白杨73的分化培养基。根据实验结果确定1/2MS基本培养基附加IBA0.4mg·L^-1、1.5％蔗糖、0.6％琼脂作为三倍体毛白杨73的生根培养基。 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：转化材料最好选用分化状态的叶片，添加乙酰丁香酮后出愈率明显增高，摇菌期间可添加100μmol·L^-1的乙酰丁香酮，将摇好的液体菌进行不同浓度稀释，以OD₆₀₀=0.4的菌液转化效果最好，叶片共培养时间为2天，在其培养阶段光培养优于暗培养，浸染时间以15-20min为宜。经实验证明，该系统对三倍体毛白杨的诱导再生率达20％。 3 根据PCR分析及Western杂交、ELISA分析的实验结果，证明Bt基因和蛋白酶抑制剂基因已整合到三倍体毛白杨基因组DNA中。通过本实验的研究，获得了转双抗虫基因的三倍体毛白杨36株，其中21株进行PCR检测，阳性率为90.5％，ELISA检测4株PCR阳性植株时毒蛋白含量测定最高表达量为0.0477％。 4 用转基因三倍体毛白杨的幼嫩叶片喂养杨扇舟蛾幼虫的虫试结果表明，转双抗虫基因三倍体毛白杨对幼虫的毒杀率明显高于对照未转基因杨树，5个编号的植株叶片被杨扇舟蛾取食后，其死亡率80％以上。说明这2个基因在细胞内均能够有效地表达，其表达产物对杨扇舟蛾幼虫具有较强的毒性。更多还原

【Abstract】 Triploid of Chinese white poplar is one of growing rapidly trees that are popularized in our country in recent years. However the quality of resisting insect has not been improved. With the enlargements of area of planting triploids of Chinese white poplar, many kinds of danger from the pest which eating leaves are more and more serious. It is important to cultivate poplar that can resist insect. Transgenosis technology has become main means to improve the quality of insect-resistant of present plant by creating new varieties.This research mainly about the transformation to the triploid of Chinese white poplar with the gene constructing two insect-resistant genes (BtCryI gene and arrowhead protease inhibitor gene API). The transgenic plant have been test tentatively by molecular biology measures and raises worms experiment . The results showed that two insect-resistant genes have been transfered into triploid of Chinese white poplar.1 Different hormone was supplemented in MS medium and their effects on shoot inducement multiplication were compared. The results showed that: MS + 6-BA 0.6mg L-1 + NAAO.lmg L-1 was the suitable combination for the inducement of buds. The selected media for rooting was: 1/2MS+IBA 0.4 mg L-1.2 Optimize studying have been done on the influencing of each factor when genes have been transformed. The result shows: the material of Transforming had better be selected the blade of the state for splitting. Produce the rate of healing to increase obviously after adding 100umol L-1 acetosyringone. In the optimum regeneration system, the optimum bacterium concentration in value of OD600 was 0.4, it had a best promoting effect on the leaf . the optimum time of infection and co-culture for leaf were 15 -20mins and 2 days respectively; Proved by the experiment the regeneration rate of Chinese white poplar of triploid reach 20% by this optimum regeneration system .3 Proved by the experiment that Bt’s gene and protease inhibitor gene have already combined in genome DNA of triploid of Chinese white poplar according to PCR analysis and western’s hybridization, ELISA. Prove gene that change over to have corresponding albumen express, toxoprotein determine most 0.0477% high person who expresses by content tentatively.4 Feeding Clostera anachoreta (Fabricius) with the young tender bladeseses of transgenosis triploid of Chinese white poplar, It shows that : Toxicity of transgenosispoplar with of a pair of genes is higher than contrast to the larva. Its death rate is more than 80% after fooding some Clostera anachoreta (Fabricius) with 5 number of this transgenosis plant. It has been proved that these 2 genes can express effectively in the cell, and what is expressed have better toxicity relatively to Clostera anachoreta (Fabricius) . It also proved that these 2 genes have different poison function in coordination to the larva by killing mechanism at the same time. It has improved the resisting worm’s function of the triploid Chinese white poplar after transfering to a pair of genes.更多还原