【作者】 袁胜亮；

【导师】 高宝嘉； 杨敏生；

【作者基本信息】 河北农业大学 ， 森林保护， 2005， 硕士

【摘要】 本研究通过饲养舞毒蛾(Lymantriadispar(Linnaeus))和杨扇舟蛾(Clostera anachoreta(Fabricius))试验筛选不同抗性的株系,并从分子生物学检测、外源基因表达规律、持续抗虫性、昆虫重要解毒酶和中肠消化酶的测定及中肠的显微观察这五个方面对转基因三倍体毛白杨进行了分析,验证转双抗基因三倍体毛白杨的抗虫性。 (1)转双抗基因三倍体毛白杨的抗虫基因的表达水平。对转双抗基因三倍体毛白杨的29个株系进行饲虫试验,其中13个株系具有较强的抗虫性,占所有参试株系的44.83%,杨扇舟蛾死亡率在80%～100%之间:10个株系抗性中等,占所有参试株系的34.48%,死亡率在60%～79%之间;6个株系抗虫性低,占所有参试株系的20.69%,杨扇舟蛾死亡率0～55%之间;与2003年饲虫结果相比,有8个株系的转基因杨的抗虫性发生变化,2003年高抗中的2号和10号变为中抗,中抗中的45号、46号和47号在2004年表现为高抗,低抗中的19号表现为高抗,24号和28号成表现为中抗。 (2)转双抗基因三倍体毛白杨的持续抗虫效应。选用高抗1号和7号,中抗10号和28号,低抗26号和16号转基因三倍体毛白杨连续饲养杨扇舟蛾的第3、4代幼虫,发现在第4代中,高抗、中抗和低抗26号株系饲养的杨扇舟蛾的死亡率相对于第三代有所上升;第三代杨扇舟蛾的蛹重明显小于Ck,第4代高抗和中抗的死亡率都是100%,低抗26号第4代的死亡率高于第3代死亡率。第3代的产卵量受到抑制,1号和7号转基因杨饲养的杨扇舟蛾的产卵量明显受到抑制,10号和28号饲养的杨扇舟蛾次之。转双抗三倍体毛白杨具有持续的抗虫效应。 (3)转双抗基因三倍体毛白杨对昆虫解毒酶及中肠消化酶的影响。杨扇舟蛾体内的羧酸酯酶、酯酶和多功能氧化酶的比活力的降低与转基因杨的抗性成反比,谷胱甘肽-S-转移酶的比活力先下降后迅速上升,然后再下降,可能与酶的性质有关。 杨扇舟蛾中肠的蛋白酶和淀粉酶也同样受到不同程度的抑制。蛋白酶活力变化较明显。1号和7号转基因杨饲养的杨扇舟蛾的中肠的蛋白酶在12h下降到最低,后上升在24h达到峰值,然后开始下降。在96h后分别为Ck的71.38%和69.66%。影响蛋白质的吸收。 (4)昆虫中肠的显微观察。转双抗基因三倍体毛白杨对幼虫的中肠有明显的影响,中肠的围食膜裂解和出现孔洞,中肠细胞变形,细胞之间界限模糊:细胞核消失;细胞质上出现空泡,空泡数量和位置异常,严重者出现溃烂现象。 (5)转基因三倍体毛白杨外源基因的PCR检测。根据PCR检测结果可知,作饲虫试验用的1号、7号、10号、28号、26号和16号转双抗基因三倍体毛白杨都含有外源抗虫基因。 关键词:三倍体毛白杨:外源基因:抗虫性:更多还原

【Abstract】 The study is aimed to screen the varies insect-resistance clones of the triploid of Chinese white poplar through feeding Lymantriadispar (Linnaeus) and Clostera anachoreta(Fabricius) test, and to validate the insect-resistance character through the test of molecule biology detection, the rule of the expression of extrinsic gene, the persistence of insect-resistance, the detoxifcation enzyme and digestive enzyme of the insects and micro-observation of the midgut.(1) There are no rules in the expression changes of the insect-resistance of the triploid of Chinese white popular. The insects were fed on 29 clones of the triploid of Chinese popular. There are 13 clones having the higher insect-resistance and account for 44.83% of all the clones. The mortality of Clostera anachoreta (Fabricius) fed on the high insect-resistance popular is among 80% and 100%. There are 10 clones having the middle insect-resistance and account for 34.48% of all the clones. The mortality of Clostera anachoreta (Fabricius) fed on the 10 clones is among 60% and 79%. There are 6 clones having the lower insect-resistance and account for 20.69% of all the clones, the death rate of Clostera anachoreta (Fabricius) fed on those clones is among 14.5% and 50%. Compared with feeding test results of 2003, there are 8 clones of insect-resistance changed. The 2 and 10 clones which were higher resistance in 2003, changed to middle insect-resistance in 2004. The middle insect-resistance, 45, 46, and 47 clones in 2003 changed to higher insect-resistance in 2004. The lower resistance, 19 clones changed to high insect-resistance, 24 and 28 clones changed to middle insect-resistance clones in 2004.(2) Transgenic triploid Chinese white poplar has lasting resisting worm’s effect. The 3 and 4 era of Clostera anachoreta (Fabricius) are fed on the higher insect-resistance 1 and 7 clones, the middle insect-resistance 10 and 28 clones and the lower insect-resistance 26 and 16 clones respectively. The death rate of the 4th generation fed on the higher and middle insect-resistance clones and 26 clones is higher than the 3rd generation. The moth weight of the 3rd generation is lower than the moth fed on CK.The death rate of the 4th generation fed on the higher and middle insect-resistance clones is 100%. Moreover, the death rate of the 4th generation feeded on 26 clones is higher than the death rate of the 3rd generation. The amount of laying of 3rd generation fed on 1 and 7 clones is largely suppressed. And the effect to larvae fed on 10 and 28 clones is lighter than fed on the 1 and 7 clones.(3) The influence of transgenic triploid Chinese white poplar to the detoxification enzymes and midgut digestive enzymes. The activities of esterase, carboxylesterase and mix-function oxidase of the Clostera anachoreta (Fabricius) larvae are inverse ratio with the insect-resistance of the transgenic triploid Chinese white popular. However, the activity of glutathione-s-transferase is improved at the first更多还原