蝎神经性毒素ANEP、AGAP的原核表达及毒力测定
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摘要
由于人类不合理的经营,常常导致森林病虫害周期性大发生,不仅给林业生产带来巨大的经济损失,而且还威胁着生态平衡。在森林害虫防治中以往多采用化学药剂,长期使用化学防治不仅导致害虫产生耐药性,而且用药量越来越大,形成恶性循环,对自然生态平衡造成严重的破坏,直接威胁着人、畜的安全,同时也增加了防治难度和资金的投入。
抗病虫基因工程的发展为防治病虫害开辟了一条新途径,对于植物的保护作用具有持续性,大大降低了病虫害的抗药性和对环境的污染,有效维护了生物多样性和生态平衡。
本文对东亚钳蝎昆虫毒素ANEP、AGAP在大肠杆菌BL-21中的最佳表达条件以及两种蝎神经性毒素实验室毒力测定进行研究,得出实验结果如下:
1.重组蛋白ANEP的理论分子量为15169.572 Da,Ala、,Cys、Gly和Thr分别占31.25%、13.02%、29.68%、26.04%;AGAP的理论分子量为15786.402 Da,其中Ala占30.3%,Cys占15.15%,Gly占28.78%,Thr占25.75%。
2.利用琼脂糖凝胶电泳对重组菌进行筛选,得到分布在标准DNA片断100bp到250bp之间的扩增基因条带,重组质粒成功转化到表达菌体中。
3.通过均匀设计实验,得出蝎神经性毒素ANEP最佳表达条件为:表达时间6h,诱导物浓度1mmol/L,菌液比例1:50,菌液OD值0.7;得出蝎神经性毒素AGAP的最佳表达条件为:表达时间6h,诱导物浓度0.5mmol/L,菌液比例1:100,菌液OD值0.5。
4.蝎神经性毒素ANEP 200倍稀释液对于分月扇舟蛾,榆紫叶甲,落叶松毛虫的校正死亡率分别为84.48%,79.31%,83.05%;蝎神经性毒素AGAP 200倍稀释液对于分月扇中蛾,榆紫叶甲,落叶松毛虫的校正死亡率分别为82.76%,68.97%,81.36%。
5.2种蝎神经性毒素重组蛋白对3种森林害虫均具有一定的杀虫效果,但ANEP杀虫效果均好于AGAP。AGAP对分月扇舟蛾的效果最佳,5种浓度的幼虫死亡率为51.72%—84.48%,对榆紫叶甲幼虫的毒力最差。
Because of the illegitimate managing,frequently lead to the heavy break of the forestry insect pests and disease,the destroy not only for forestry production,but also for the ecological balance.From the year 1996 to 2000,the average square of insect pests and disease is about 7.3 million hectares,and it is about 8.67 million hectares in the year 2001,more and the most heavy break squares are keep for 60%,is remain higher lever.It was used chemical reagent for the prevention and cure of insect pests and disease,it is not only lead to the insect chemical-resisting,but also a vicious circle what more and more volume of use bring the serious destroy for the balance of nature,also threaten the safe of human and animal,bad for the grow of forestry,and bring more difficulties to prevent and cure of insect pests and disease,the finance of prevent and cure is increasing.
The development of insect and disease resistant genetic engineering provides another way for the prevention of plant diseases and insect pest,Compare with the traditional methods, The technique has such advantages as follows:strong selectivity,less investment and take effect quickly.Besides,the protection of plant continuity and integrality,The resistance of plant diseases and insect pests and environmental pollution have debased greatly.The multiform of life-form and ecological balance maintenance effectively. In this thesis,it use the even design for the expression of Scorpion Toxin ANEP and AGAP in E.coli BL-21 to do experiment for the best expression conditions of expression and gain the result of biologic insecticide experiment.AS the result:
(1)With the experiment,we get the protein volume under the best conditions.
The best expression condition for Scorpion Toxin ANEP is:expression time 6h,1mmol/L, 1:50,OD0.7.
The best expression condition for Scorpion Toxin ANEP is:expression time 6h, 0.5mmol/L,1:100,OD0.5.
(2)With the biologic insecticide experiment,we can get the result as:
The best death rate of Scorpion Toxin ANEP with Clostera anastomosis,Ambrostoma quadriimpressum and Dendrolin us superans Buthler after 7 days are 84.48%,79.31%, 83.05%.
The best death rate of Scorpion Toxin AGAP with Clostera anastomosis,Ambrostoma quadriimpressum and Dendrolin us superans Buthler after 7 days are 82.76%,68.97%, 81.36%.
抗病虫基因工程的发展为防治病虫害开辟了一条新途径,对于植物的保护作用具有持续性,大大降低了病虫害的抗药性和对环境的污染,有效维护了生物多样性和生态平衡。
本文对东亚钳蝎昆虫毒素ANEP、AGAP在大肠杆菌BL-21中的最佳表达条件以及两种蝎神经性毒素实验室毒力测定进行研究,得出实验结果如下:
1.重组蛋白ANEP的理论分子量为15169.572 Da,Ala、,Cys、Gly和Thr分别占31.25%、13.02%、29.68%、26.04%;AGAP的理论分子量为15786.402 Da,其中Ala占30.3%,Cys占15.15%,Gly占28.78%,Thr占25.75%。
2.利用琼脂糖凝胶电泳对重组菌进行筛选,得到分布在标准DNA片断100bp到250bp之间的扩增基因条带,重组质粒成功转化到表达菌体中。
3.通过均匀设计实验,得出蝎神经性毒素ANEP最佳表达条件为:表达时间6h,诱导物浓度1mmol/L,菌液比例1:50,菌液OD值0.7;得出蝎神经性毒素AGAP的最佳表达条件为:表达时间6h,诱导物浓度0.5mmol/L,菌液比例1:100,菌液OD值0.5。
4.蝎神经性毒素ANEP 200倍稀释液对于分月扇舟蛾,榆紫叶甲,落叶松毛虫的校正死亡率分别为84.48%,79.31%,83.05%;蝎神经性毒素AGAP 200倍稀释液对于分月扇中蛾,榆紫叶甲,落叶松毛虫的校正死亡率分别为82.76%,68.97%,81.36%。
5.2种蝎神经性毒素重组蛋白对3种森林害虫均具有一定的杀虫效果,但ANEP杀虫效果均好于AGAP。AGAP对分月扇舟蛾的效果最佳,5种浓度的幼虫死亡率为51.72%—84.48%,对榆紫叶甲幼虫的毒力最差。
Because of the illegitimate managing,frequently lead to the heavy break of the forestry insect pests and disease,the destroy not only for forestry production,but also for the ecological balance.From the year 1996 to 2000,the average square of insect pests and disease is about 7.3 million hectares,and it is about 8.67 million hectares in the year 2001,more and the most heavy break squares are keep for 60%,is remain higher lever.It was used chemical reagent for the prevention and cure of insect pests and disease,it is not only lead to the insect chemical-resisting,but also a vicious circle what more and more volume of use bring the serious destroy for the balance of nature,also threaten the safe of human and animal,bad for the grow of forestry,and bring more difficulties to prevent and cure of insect pests and disease,the finance of prevent and cure is increasing.
The development of insect and disease resistant genetic engineering provides another way for the prevention of plant diseases and insect pest,Compare with the traditional methods, The technique has such advantages as follows:strong selectivity,less investment and take effect quickly.Besides,the protection of plant continuity and integrality,The resistance of plant diseases and insect pests and environmental pollution have debased greatly.The multiform of life-form and ecological balance maintenance effectively. In this thesis,it use the even design for the expression of Scorpion Toxin ANEP and AGAP in E.coli BL-21 to do experiment for the best expression conditions of expression and gain the result of biologic insecticide experiment.AS the result:
(1)With the experiment,we get the protein volume under the best conditions.
The best expression condition for Scorpion Toxin ANEP is:expression time 6h,1mmol/L, 1:50,OD0.7.
The best expression condition for Scorpion Toxin ANEP is:expression time 6h, 0.5mmol/L,1:100,OD0.5.
(2)With the biologic insecticide experiment,we can get the result as:
The best death rate of Scorpion Toxin ANEP with Clostera anastomosis,Ambrostoma quadriimpressum and Dendrolin us superans Buthler after 7 days are 84.48%,79.31%, 83.05%.
The best death rate of Scorpion Toxin AGAP with Clostera anastomosis,Ambrostoma quadriimpressum and Dendrolin us superans Buthler after 7 days are 82.76%,68.97%, 81.36%.
引文
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