拮抗细菌的筛选鉴定及其环脂肽合成相关基因检测

发布时间：2018-03-04 07:23

本文选题：拮抗细菌　切入点：芽胞杆菌　出处：《北京农学院》2016年硕士论文　论文类型：学位论文

【摘要】：随着人们对食品安全和改善生态环境要求的不断提高,生物防治也越来越受到重视,并逐渐成为研究和应用的热点。本研究对实验室分离得到的拮抗细菌进行了初步筛选鉴定和抑菌谱检测,并PCR验证了各拮抗细菌中的4种环脂肽合成相关基因,以对拮抗细菌的拮抗机理进行初步判定。本研究主要结果如下：1、从北极采回土壤样品中共分离出12株细菌,平板对峙法对其进行了拮抗活性试验的初步筛选,结果表明,只有X1-2对草莓褐色轮斑病菌(Sphaeronaemella fragariae) CMB-2具有一定的拮抗活性,其抑菌带的宽度达17 mm,而其他11株细菌均未产生明显的抑菌带。2、对实验室保存的8株拮抗细菌以及从北极土壤样品中筛选得到的X1-2进行了16S rDNA、gyrA、gyrB基因的分子生物学鉴定,结果表明：在9株被鉴定的拮抗细菌中有5株解淀粉芽胞杆菌(Bacillus amyloliquefaciens),分别为BJ-6、BJ-7、BJ-8、22#口33#：2株枯草芽胞杆菌(Bacillus subtilis) BJ-9和X1-2；1株嗜麦芽寡养单胞菌(Stenotrophomonas maltophilid) 1#和1株苏云金芽胞杆菌(Bacillus thuringiensis) BT。3、测定了9株拮抗细菌对植物病原菌的抑菌活性,结果表明：B. amyloliquefaciens BJ-6、BJ-7、BJ-8、22#、33#抑菌谱较广,且抑菌活性强；S. maltophilia 1#次之；而B. subtilis BJ-9、X1-2和5. thuringiensis BT的抑菌谱较窄,抑菌活性相对较弱。4、对9株细菌进行了4种环脂肽合成基因(fenD、bacD、ituA、sfp)的PCR检测,结果表明B.amyloliquefaciens 中BJ-6、BJ-8、22#、33#菌株中均含有4中所检测的基因,而5. amyloliquefaciens BJ-7中未检测到表面活性素基因；5. maltophilia 1#中未检测到表面活性素基因sfp和丰原素基因fenD;而四种所检测的基因均未扩增到的为B. subtilis BJ-9和X1-2和苏云金芽胞杆菌BT。5、利用重叠PCR技术将丰原素合成基因fenD上下游连接在一起,连接在温敏质粒pKSV7上构建fenD基因缺失载体；然后利用同源交换的方法对B. amyloliquefaciens BJ-6的丰原素合成相关基因进行缺失突变。由于电激转化率低,以及基因重组率低的原因,结果并未筛选出突变菌株。
[Abstract]:With the increasing demands on food safety and improving ecological environment, biological control has been paid more and more attention. In this study, the antagonistic bacteria isolated in the laboratory were screened and identified, and the bacteriostasis spectrum was detected, and PCR confirmed four genes related to the synthesis of cyclopeptide in the antagonistic bacteria. The main results of this study are as follows: 1. 12 strains of bacteria were isolated from soil samples collected from the Arctic. Only X1-2 had a certain antagonistic activity against CMB-2 of Sphaeronaemella fragariaeae. The width of the inhibition band was 17 mm, but none of the other 11 strains produced obvious inhibition band. The molecular biological identification of 8 antagonistic bacteria preserved in laboratory and X1-2 gene selected from Arctic soil samples was carried out. The results showed that among the 9 identified antagonistic bacteria, 5 were Bacillus amyloliquefaciens1#, which were BJ-6, BJ-7, BJ-8, 2, #33: 2, Bacillus subtilisi, and X1-21, Stenotrophomonas maltophilid1# and Bacillus thuringiensis, respectively. The inhibitory activity of 9 antagonistic bacteria against plant pathogens was determined. The results showed that the bacteriostatic spectrum of amyloliquefaciens BJ-6, BJ-7, BJ-822, #an3 was broad, and the bacteriostatic activity was higher than that of S. maltophilia 1#, while that of BJ-9 X1-2 and 5. thuringiensis BT was narrower and weaker than that of thuringiensis BT. PCR was used to detect the four cyclopeptide synthesizer genes, fendacidin DituAfp, from 9 strains of bacteria, and the results showed that the inhibition spectrum of BJ-6 and BJ-7 BJ-8 was higher than that of thuringiensis BJ-7BJ-8, and the antibacterial activity was higher than that of S.#en1# 1#, while the inhibitory spectrum of BJ-9 X1-2 and 5. thuringiensis BT was relatively weak. The results showed that the BJ-6, BJ-8J22, #ing-33# strains of B.amyloliquefaciens contained 4 genes, However, no surface active gene was detected in amyloliquefaciens BJ-7. Sfp and fenDin genes were not detected in maltophilia 1#, while B.#en4# BJ-9 and X1-2 and Bacillus thuringiensis BT.5 were not amplified from the four detected genes. The gene fenD was linked together by overlapping PCR technique. The deletion vector of fenD gene was constructed by ligating into the thermo-sensitive plasmid pKSV7, and the deletion mutation of the gene related to the oncogene synthesis of B. amyloliquefaciens BJ-6 was carried out by using homology exchange method. Due to the low conversion rate of electric excitation and the low rate of gene recombination, The mutant strain was not screened out.
【学位授予单位】：北京农学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476

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