轉(zhuǎn)Mad1基因球孢白僵菌工程菌株的構(gòu)建及鑒定
發(fā)布時間：2019-04-01 12:49
【摘要】：在全球關(guān)注綠色無公害農(nóng)業(yè)的大前提下,微生物農(nóng)藥得到了廣泛的關(guān)注和長足的發(fā)展,雖然存在著寄主范圍廣、致病力強(qiáng)、對人畜無害等優(yōu)點(diǎn),不過缺點(diǎn)也是顯而易見的,如致病潛伏期長,殺蟲時效性較差、對環(huán)境條件要求高等缺點(diǎn),使得微生物殺蟲劑的推廣仍然受到限制。因此利用基因工程等技術(shù)手段改造真菌,獲得具有更高殺蟲力并適應(yīng)市場需求的微生物殺蟲劑將具有重要的應(yīng)用價值。球孢白僵菌(Beauveria bassiana)作為重要的昆蟲病原真菌在害蟲的生物防治中發(fā)揮著重要作用。目前許多球孢白僵菌的基因工程研究主要集中在毒力直接相關(guān)基因的轉(zhuǎn)化和表達(dá),如蛋白酶基因、幾丁質(zhì)酶基因、毒素基因等,存在著一定的生物安全性問題,本研究所導(dǎo)入的外源基因來自于金龜子綠僵菌(Metarhizium anisopliae),并且該基因非直接毒素基因,而是通過空氣表達(dá)增加蟲生真菌黏著性,促進(jìn)孢子對昆蟲體壁的吸附,從而增加毒力,縮短作用時間,對未來白僵菌基因工程菌株的選育提供了材料。本研究克隆了來源于生防真菌綠僵菌的黏著蛋白基因Mad1,構(gòu)建到遺傳轉(zhuǎn)化載體上。采用溶壁酶和β-巰基乙醇裂解白僵菌細(xì)胞壁,得到的原生質(zhì)體濃度達(dá)到108sp/mL,再生培養(yǎng)基上培養(yǎng)經(jīng)計算其再生率為15%。使用抗殺菌劑萎銹靈基因Carboxin做為抗性篩選標(biāo)記,經(jīng)抗性濃度篩選確定其抗性終濃度為180ng/mL。利用PEG介導(dǎo)的原生質(zhì)體遺傳轉(zhuǎn)化體系導(dǎo)入受體球孢白僵菌菌株,獲得轉(zhuǎn)化子,經(jīng)過繼代培養(yǎng)及分子檢測,獲得遺傳穩(wěn)定的基因工程菌株。利用蝗蟲內(nèi)翅及洋蔥表皮進(jìn)行吸附試驗(yàn)進(jìn)行了工程菌株的生物學(xué)功能鑒定。研究結(jié)果表明,野生型和工程菌的分生孢子對于洋蔥表皮的吸附量并無明顯差異,而工程菌的分生孢子對蝗蟲翅膀的吸附則顯著高于野生型,表明Mad1基因的導(dǎo)入能夠增加球孢白僵菌對昆蟲組織的吸附,而對植物表皮組織不存在吸附能力;對亞洲玉米螟幼蟲室內(nèi)毒力測定結(jié)果表明轉(zhuǎn)化后的工程菌株LT50值(3.278±0.346天)要比野生型(4.972±0.147天)縮短1天。殺蟲率方面轉(zhuǎn)化后的工程菌株殺蟲校正死亡率在第七天時達(dá)到82.88%,而野生菌株為69.27%,殺蟲效率顯著提高。
[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 鹵0.346 days) was 1 day shorter than that of wild type strain (4.972 鹵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.
【學(xué)位授予單位】：哈爾濱師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S476.12
本文編號：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 鹵0.346 days) was 1 day shorter than that of wild type strain (4.972 鹵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.
【學(xué)位授予單位】：哈爾濱師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S476.12
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 付志堅,陳建新,付麗君;白僵菌對昆蟲的致病機(jī)理研究綜述[J];武夷科學(xué);2000年00期
，本文編號：2451569
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