【作者】 吕丁丁；

【导师】 李增智； 王成树；

【作者基本信息】 安徽农业大学 ， 森林保护学， 2008， 硕士

【摘要】 随着社会经济的发展和人们环保意识的提高,环境友好型生物源杀虫剂的应用受到越来越高的重视。利用昆虫病原真菌加工而成的真菌杀虫剂的应用及基础研究得到了长足的发展,但由于存在击倒昆虫时间较长、对环境条件要求高等缺点,真菌杀虫剂的广泛应用受到限制。因此利用基因工程和细胞工程等技术提高菌株毒力,遗传改造获得适合市场需要的真菌杀虫剂具有重要的应用价值。球孢白僵菌（Beauveriabassiana）作为重要的昆虫病原真菌在害虫的生物防治中发挥着重要作用。本文以球孢白僵菌作为出发菌株,同时引入双价毒力基因,即降解昆虫体壁的类枯草杆菌蛋白酶基因Pr1A和北非蝎（Androctonus australis）昆虫特异性神经毒素多肽基因AaIT,构建了高毒力双价工程菌株。为了获得双价工程菌株,本实验首先构建了含有不同筛选标记和目的基因的载体pGPS3Ben-Pr1A和pGPET1。并且通过对分生孢子的抑制性实验,筛选出两种抗性药对球孢白僵菌的最佳抑制浓度,其中草胺磷对球孢白僵菌的最佳抑制浓度为100μg/ml,苯菌灵对球孢白僵菌的最佳抑制浓度为2μg/ml。本实验选用了两种不同的遗传转化方法对球孢白僵菌进行遗传改造,获得了一价工程菌株Bb13T和Bb202T,及二价工程菌株Bb13Tpr1A。RT-PCR和Western blot结果证明,Pr1A和AaIT均在重组菌株中组成性转录和表达。PR1A蛋白酶活性测定结果表明,超表达PR1A的二价工程菌株的酶活相对于野生型菌株得到了极大的提高。并且通过初步的注射虫体实验,筛选出毒力相对较高的一价工程菌株Bb13T-4-1、Bb13T-10-3和Bb202T-7,和二价工程菌株Bb13TPr1A-1、Bb13TPr1A-2和Bb13TPr1A-3。对松墨天牛（Monochamus alternatus）的生物测定结果表明,超量表达AAIT的一价工程菌株Bb202T-7提高了杀虫效率约11.1%。使用大蜡螟（Galleria mellonella）和马尾松毛虫（Dendrolimus punctatus）进行生物测定的结果同样显示,超量表达AaIT单价基因及AaIT和pr1A双价基因均能显著地提高重组菌株的毒力。用大蜡螟做生测试虫,在孢子浓度为1×10⁷个/ml时,与野生型菌株Bb13相比,超量表达AaIT的重组菌株Bb13T的致死中时(LT₅₀)平均缩短了24.4%,同时超量表达AaIT和pr1A双价基因的Bb13TPr1A的LT₅₀平均缩短了20.9%。马尾松毛虫的生物测定中,在孢子浓度为1×10⁷个/ml时,与野生型菌株Bb13相比,超量表达AaIT基因的重组菌株Bb13T的LT₅₀平均缩短了40%,其致死中浓度(LC₅₀)降低了约15倍的剂量;超量表达AaIT和Pr1A双价基因的Bb13Tpr1A的LT₅₀平均缩短了36.7%,同时Bb13TPr1A的LC₅₀也降低了约8倍剂量。蛋白酶抑制实验及Western blot结果证明PR1A对AAIT有降解作用。并且生物测定结果表明,二价菌株的杀虫效率低于一价菌株的杀虫效率,更充分证明了PR1A对AAIT具有降解作用。因此在以后构建多价工程菌株时,首先要充分分析蛋白之间的相互作用以选出最佳的基因组合。更多还原

【Abstract】 Along with the development of economy and improvement of people’s awareness of environmental protection,the environmentally friendly mycoinsecticides have received great attention.However,all fungal biocontrol agents inevitably experience a latent period for infection development in host hemocoel and always kill insects more slowly than chemical insecticides.This disadvantage has often discouraged the use of fungal formulations in insect pest control.In order to increase mycoinsecticide products in market, it is urgently needed to improve the strain performance by genetic engineering.The entomopathogenic fungus Beauveria bassiana plays a significant role in the bio-control against pests.In this paper,Beauveria bassiana was used for genetical modification with an insect-specific scorpion neurotoxin AaIT and an insect cuticle degradation protease Pr1A by two step transformations using different selective markers.In order to obtain dual engineering strains,the plasmids pGPS3Ben-Pr1A and pGPET1 were constructed with different selective markers and virulent genes.The optimal inhibitory concentrations were selected by drug resistance assays with conidia.The best inhibitory concentration for Glufosinate Ammonium was 100ug/ml,and the best inhibitory concentration for Benomyl was 2ug/ml.Through two different genetic transformation methods,the singly transgenic isolates of Bb13T,Bb202T and the double transgenic strain Bb13TPr1A were acquired.RT-PCR and Western blot results show that pr1A and AaIT are successfully transcribed and expressed by recombinant strains.PR1A activity assays showed that the PR1A activity of double transformants had been greatly improved in comparison to the wild-type strains.Through the initial injection of Bornbyx mori larvae, the transgenic isolates with relatively high virulence were chose for further analysis.The selected AaIT singly transgenic isolates were Bb13T-4-1,Bb13T-10-3 and Bb202T-7.And the double transgenic isolates were Bb13TPr1A-1,Bb13TPr1A-2 and Bb13TPSr1A-3.To evaluate the insecticidal efficacy of transgenic isolates,the wide type Bb202 and the AaIT singly transgenic transformant Bb202T-7 were bioassayed against Monochamus alternatus.The data revealed that the singly transgenic transformant received an improved pathogenic effect by reducing the time of insect death up to 11.1%.At the same time,the wild type and the transformants were bioassayed against the larvae of Masson’s pine caterpillar Dendrolimus punctatus and the wax moth Galleria mellonella,respectively.As compared with the wild type,engineered isolates took fewer spores to kill 50%of pine caterpillars,i.e.15-fold less for AaIT single transformant Bb13T and 8-fold less for double transformant Bb13TPr1A,respectively.The median lethal time for Bb13T and Bb13TPr1A were reduced by 40%and 36.7%,respectively against D.punctatus and 24.4%and 20.9 %,respectively against G.mellonella.It is evident in this study that AAIT could be degraded by protease PR1A when both present in samples,recommending that protein interactions should be evaluated before future fungal transformation with multiple genes.更多还原