转Mad1基因球孢白僵菌工程菌株的构建及鉴定
发布时间：2019-04-01 12:49
【摘要】：在全球关注绿色无公害农业的大前提下,微生物农药得到了广泛的关注和长足的发展,虽然存在着寄主范围广、致病力强、对人畜无害等优点,不过缺点也是显而易见的,如致病潜伏期长,杀虫时效性较差、对环境条件要求高等缺点,使得微生物杀虫剂的推广仍然受到限制。因此利用基因工程等技术手段改造真菌,获得具有更高杀虫力并适应市场需求的微生物杀虫剂将具有重要的应用价值。球孢白僵菌(Beauveria bassiana)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。目前许多球孢白僵菌的基因工程研究主要集中在毒力直接相关基因的转化和表达,如蛋白酶基因、几丁质酶基因、毒素基因等,存在着一定的生物安全性问题,本研究所导入的外源基因来自于金龟子绿僵菌(Metarhizium anisopliae),并且该基因非直接毒素基因,而是通过空气表达增加虫生真菌黏着性,促进孢子对昆虫体壁的吸附,从而增加毒力,缩短作用时间,对未来白僵菌基因工程菌株的选育提供了材料。本研究克隆了来源于生防真菌绿僵菌的黏着蛋白基因Mad1,构建到遗传转化载体上。采用溶壁酶和β-巯基乙醇裂解白僵菌细胞壁,得到的原生质体浓度达到108sp/mL,再生培养基上培养经计算其再生率为15%。使用抗杀菌剂萎锈灵基因Carboxin做为抗性筛选标记,经抗性浓度筛选确定其抗性终浓度为180ng/mL。利用PEG介导的原生质体遗传转化体系导入受体球孢白僵菌菌株,获得转化子,经过继代培养及分子检测,获得遗传稳定的基因工程菌株。利用蝗虫内翅及洋葱表皮进行吸附试验进行了工程菌株的生物学功能鉴定。研究结果表明,野生型和工程菌的分生孢子对于洋葱表皮的吸附量并无明显差异,而工程菌的分生孢子对蝗虫翅膀的吸附则显著高于野生型,表明Mad1基因的导入能够增加球孢白僵菌对昆虫组织的吸附,而对植物表皮组织不存在吸附能力;对亚洲玉米螟幼虫室内毒力测定结果表明转化后的工程菌株LT50值(3.278±0.346天)要比野生型(4.972±0.147天)缩短1天。杀虫率方面转化后的工程菌株杀虫校正死亡率在第七天时达到82.88%,而野生菌株为69.27%,杀虫效率显著提高。
[Abstract]:Under the premise of global concern about green and pollution-free agriculture, microbial pesticides have received extensive attention and considerable development. Although there are many advantages such as wide host range, strong pathogenicity and harmless to humans and animals, but the disadvantages are also obvious. If the latent period of disease is long, the time of killing insects is poor, and the requirement of environmental conditions is high, the popularization of microbial insecticides is still limited. Therefore, it is of great value to use genetic engineering and other techniques to reform fungi and obtain microbial insecticides which have higher insecticidal power and adapt to the market demand. As an important entomopathogenic fungus, Beauveria bassiana (Beauveria bassiana) plays an important role in the biological control of insect pests. At present, many genetic engineering studies of Beauveria bassiana mainly focus on the transformation and expression of virulence-related genes, such as protease gene, chitinase gene, toxin gene, and so on. There are some biological safety problems, such as protease gene, chitinase gene, toxin gene and so on. The exogenous gene from Metarhizium anisopliae (Metarhizium anisopliae), and the non-direct toxin gene of the gene were introduced in this study, but increased the adhesion of entomogenous fungi through air expression, and promoted the adsorption of spores to insect body walls, thus increasing the virulence. Shortening the action time provides materials for the breeding of Beauveria bassiana genetic engineering strains in the future. In this study, we cloned the adhesive protein gene Mad1, from Metarhizium anisopliae, a biocontrol fungus, and constructed it into a genetic transformation vector. The cell wall of Beauveria bassiana was lysed by lysozyme and 尾-mercaptoethanol, and the protoplast concentration reached 108 sp / mL. The regeneration rate was calculated to be 15% on regeneration medium. Carboxin gene was used as a marker for resistance screening, and the final resistance concentration was determined to be 180 ng / ml 路L-1 by screening the resistance concentration. The protoplast genetic transformation system mediated by PEG was introduced into Beauveria bassiana strain, and the transformants were obtained. After subculture and molecular detection, the genetically stable genetic engineering strains were obtained. The biological function of the engineering strain was identified by adsorption test of locust inner wing and onion epidermis. The results showed that there was no significant difference in the adsorption capacity of conidia between wild type and engineering bacteria to onion epidermis, while the adsorption of conidia of engineering bacteria to grasshopper wings was significantly higher than that of wild type. The results showed that the introduction of Mad1 gene could increase the adsorption of insect tissues by Beauveria bassiana, but there was no adsorption ability to plant epidermis. The virulence test of Asian corn borer larvae showed that the LT50 value of the transformed strain (3.278 卤0.346 days) was 1 day shorter than that of wild type strain (4.972 卤0.147 days). The corrected mortality rate of the transformed engineering strains reached 82.88% on the seventh day, while 69.27% of the wild strains, which significantly improved the insecticidal efficiency.
【学位授予单位】：哈尔滨师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.12
本文编号：2451569
[Abstract]:Under the premise of global concern about green and pollution-free agriculture, microbial pesticides have received extensive attention and considerable development. Although there are many advantages such as wide host range, strong pathogenicity and harmless to humans and animals, but the disadvantages are also obvious. If the latent period of disease is long, the time of killing insects is poor, and the requirement of environmental conditions is high, the popularization of microbial insecticides is still limited. Therefore, it is of great value to use genetic engineering and other techniques to reform fungi and obtain microbial insecticides which have higher insecticidal power and adapt to the market demand. As an important entomopathogenic fungus, Beauveria bassiana (Beauveria bassiana) plays an important role in the biological control of insect pests. At present, many genetic engineering studies of Beauveria bassiana mainly focus on the transformation and expression of virulence-related genes, such as protease gene, chitinase gene, toxin gene, and so on. There are some biological safety problems, such as protease gene, chitinase gene, toxin gene and so on. The exogenous gene from Metarhizium anisopliae (Metarhizium anisopliae), and the non-direct toxin gene of the gene were introduced in this study, but increased the adhesion of entomogenous fungi through air expression, and promoted the adsorption of spores to insect body walls, thus increasing the virulence. Shortening the action time provides materials for the breeding of Beauveria bassiana genetic engineering strains in the future. In this study, we cloned the adhesive protein gene Mad1, from Metarhizium anisopliae, a biocontrol fungus, and constructed it into a genetic transformation vector. The cell wall of Beauveria bassiana was lysed by lysozyme and 尾-mercaptoethanol, and the protoplast concentration reached 108 sp / mL. The regeneration rate was calculated to be 15% on regeneration medium. Carboxin gene was used as a marker for resistance screening, and the final resistance concentration was determined to be 180 ng / ml 路L-1 by screening the resistance concentration. The protoplast genetic transformation system mediated by PEG was introduced into Beauveria bassiana strain, and the transformants were obtained. After subculture and molecular detection, the genetically stable genetic engineering strains were obtained. The biological function of the engineering strain was identified by adsorption test of locust inner wing and onion epidermis. The results showed that there was no significant difference in the adsorption capacity of conidia between wild type and engineering bacteria to onion epidermis, while the adsorption of conidia of engineering bacteria to grasshopper wings was significantly higher than that of wild type. The results showed that the introduction of Mad1 gene could increase the adsorption of insect tissues by Beauveria bassiana, but there was no adsorption ability to plant epidermis. The virulence test of Asian corn borer larvae showed that the LT50 value of the transformed strain (3.278 卤0.346 days) was 1 day shorter than that of wild type strain (4.972 卤0.147 days). The corrected mortality rate of the transformed engineering strains reached 82.88% on the seventh day, while 69.27% of the wild strains, which significantly improved the insecticidal efficiency.
【学位授予单位】：哈尔滨师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.12
【参考文献】
相关期刊论文 前1条
1 付志坚,陈建新,付丽君;白僵菌对昆虫的致病机理研究综述[J];武夷科学;2000年00期
，本文编号：2451569
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