解淀粉芽孢桿菌PEBA20群體感應(yīng)基因簇的克隆及相關(guān)特性研究
發(fā)布時(shí)間：2018-08-04 10:04
【摘要】：本研究對(duì)解淀粉芽孢桿菌PEBA20菌株的群體感應(yīng)相關(guān)基因進(jìn)行基因克隆并作出生物信息學(xué)分析,以期了解其基因結(jié)構(gòu)特征;利用同源重組法構(gòu)建了comA基因缺失突變株進(jìn)而驗(yàn)證該基因及群體感應(yīng)系統(tǒng)對(duì)生防活性、生物膜形成的影響。主要研究結(jié)果如下:1.從Bacillus amyloliquefaciens PEBA20中克隆了群體感應(yīng)系統(tǒng)基因comQXPA,通過(guò)序列比對(duì)分析發(fā)現(xiàn),comA基因的保守性是最高的,其次是comP,comX保守性最低。對(duì)蛋白功能進(jìn)行預(yù)測(cè)發(fā)現(xiàn)預(yù)期功能相適應(yīng)的結(jié)構(gòu)。2.構(gòu)建了comA基因缺失突變株,comA基因同源前臂和同源后臂的連接質(zhì)粒comAF-comAB與自殺質(zhì)粒pk18mobsacB經(jīng)酶切、T4連接酶連接得到重組質(zhì)粒comAFcomAB-pk18mobsacB。將構(gòu)建得到的重組質(zhì)粒經(jīng)電轉(zhuǎn)化方法轉(zhuǎn)化入B.amyloliquefaciens PEBA20感受態(tài)細(xì)胞中,采用卡那篩選法及繼代培養(yǎng)大量篩選獲得comA基因缺失的PEBA20突變株。以缺失株DNA為模板進(jìn)行PCR篩選陽(yáng)性克隆,并通過(guò)測(cè)序進(jìn)一步確定獲得comA基因缺失突變株。3.對(duì)comA基因缺失突變株的生物膜表型進(jìn)行檢測(cè),發(fā)現(xiàn)固氣界面comA基因缺失突變株在NB平板上初期為白色平滑圓形的小菌落,表面無(wú)褶皺。之后菌落外圍呈放射狀,內(nèi)部呈現(xiàn)一個(gè)圓圈,圓圈邊緣界限明顯呈白色;隨著時(shí)間的推移菌落邊緣擴(kuò)大速度明顯高于內(nèi)部小圓圈的速度;后期菌落不再擴(kuò)大,菌落邊緣分化程度明顯,白色邊緣與透明邊緣交替出現(xiàn);氣液界面12h后已出現(xiàn)渾濁,至24h時(shí)已形成一層平整的薄薄的生物膜,36h生物膜已出現(xiàn)破裂降解,生物膜不完整,底部出現(xiàn)菌體沉淀;隨著時(shí)間的推移生物膜逐漸降解完全菌體在底部沉淀。表明comA基因影響了生物膜形成及其復(fù)雜程度。4.檢測(cè)B.amyloliquefaciens PEBA20野生株和comA基因缺失突變株對(duì)四種真菌的抑菌活性。結(jié)果顯示,與野生株相比,comA基因缺失突變株對(duì)四種指示真菌抑菌效果均下降。這表明comA基因的缺失突變影響了生防細(xì)菌對(duì)指示真菌的抑菌活性作用。
[Abstract]:In this study, the genes related to population induction of Bacillus amylolyticus PEBA20 strain were cloned and bioinformatics analysis was made in order to understand the structural characteristics of the genes. The comA gene deletion mutant was constructed by homologous recombination method to verify the effect of the gene and the population sensing system on biocontrol activity and biofilm formation. The main results are as follows: 1. The population sensing system gene comQXPA was cloned from Bacillus amyloliquefaciens PEBA20, and the sequence alignment analysis showed that the conserved character of comcomA gene was the highest, followed by the lowest conservation of comcomX gene. The function of protein was predicted and the structure of expected function was found. 2. 2. The ligation plasmid comAF-comAB of the homologous forearm and hind arm of the comA gene deletion mutant was constructed and ligated with the suicide plasmid pk18mobsacB to obtain the recombinant plasmid comAFcomAB-pk18mobsacB. The constructed recombinant plasmid was transformed into B.amyloliquefaciens PEBA20 receptive cells by electrotransformation. A large number of PEBA20 mutants with comA gene deletion were obtained by Karna screening and subculture. The deletion strain DNA was used as template to screen the positive clones of PCR, and the comA gene deletion mutant. 3 was further confirmed by sequencing. The biofilm phenotype of the comA gene deletion mutant was detected. It was found that the comA gene deletion mutant on the solid gas interface was a white, smooth, circular colony at the initial stage of the NB plate and had no fold on the surface. The outer circle of the colony was radial, the inner circle appeared a circle, the boundary of the circle was obviously white; over time, the expanding speed of the colony edge was obviously higher than that of the inner small circle; the later colony no longer expanded. The differentiation degree of colony edge was obvious, the white edge and transparent edge appeared alternately, the gas-liquid interface appeared turbidity after 12 hours, and by 24 hours, a thin and flat biofilm had been formed, and the biofilm had broken down and degraded, and the biofilm was incomplete. At the bottom, the bacteria were precipitated, and the biofilm gradually degraded at the bottom. The results showed that comA gene affected biofilm formation and its complexity. The antimicrobial activity of B.amyloliquefaciens PEBA20 wild strain and comA gene deletion mutant against four fungi was detected. The results showed that compared with wild strains, the inhibitory effect of the mutant on four indicator fungi was decreased. This indicated that the deletion of comA gene affected the bacteriostatic activity of biocontrol bacteria against indicator fungi.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：Q78;S476.1
本文編號(hào)：2163531
[Abstract]:In this study, the genes related to population induction of Bacillus amylolyticus PEBA20 strain were cloned and bioinformatics analysis was made in order to understand the structural characteristics of the genes. The comA gene deletion mutant was constructed by homologous recombination method to verify the effect of the gene and the population sensing system on biocontrol activity and biofilm formation. The main results are as follows: 1. The population sensing system gene comQXPA was cloned from Bacillus amyloliquefaciens PEBA20, and the sequence alignment analysis showed that the conserved character of comcomA gene was the highest, followed by the lowest conservation of comcomX gene. The function of protein was predicted and the structure of expected function was found. 2. 2. The ligation plasmid comAF-comAB of the homologous forearm and hind arm of the comA gene deletion mutant was constructed and ligated with the suicide plasmid pk18mobsacB to obtain the recombinant plasmid comAFcomAB-pk18mobsacB. The constructed recombinant plasmid was transformed into B.amyloliquefaciens PEBA20 receptive cells by electrotransformation. A large number of PEBA20 mutants with comA gene deletion were obtained by Karna screening and subculture. The deletion strain DNA was used as template to screen the positive clones of PCR, and the comA gene deletion mutant. 3 was further confirmed by sequencing. The biofilm phenotype of the comA gene deletion mutant was detected. It was found that the comA gene deletion mutant on the solid gas interface was a white, smooth, circular colony at the initial stage of the NB plate and had no fold on the surface. The outer circle of the colony was radial, the inner circle appeared a circle, the boundary of the circle was obviously white; over time, the expanding speed of the colony edge was obviously higher than that of the inner small circle; the later colony no longer expanded. The differentiation degree of colony edge was obvious, the white edge and transparent edge appeared alternately, the gas-liquid interface appeared turbidity after 12 hours, and by 24 hours, a thin and flat biofilm had been formed, and the biofilm had broken down and degraded, and the biofilm was incomplete. At the bottom, the bacteria were precipitated, and the biofilm gradually degraded at the bottom. The results showed that comA gene affected biofilm formation and its complexity. The antimicrobial activity of B.amyloliquefaciens PEBA20 wild strain and comA gene deletion mutant against four fungi was detected. The results showed that compared with wild strains, the inhibitory effect of the mutant on four indicator fungi was decreased. This indicated that the deletion of comA gene affected the bacteriostatic activity of biocontrol bacteria against indicator fungi.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：Q78;S476.1
【共引文獻(xiàn)】
相關(guān)期刊論文 前1條
1 賈翠麗;張華偉;王斌斌;朱宏吉;喬建軍;;革蘭氏陽(yáng)性菌自然感受態(tài)生理機(jī)制的研究進(jìn)展[J];中國(guó)生物工程雜志;2015年06期
，本文編號(hào)：2163531
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