基于NRPS基因筛选鉴定产生环脂肽葡萄附生细菌的研究
发布时间：2018-11-02 18:39
【摘要】：本研究采用平板稀释法和斜面分离纯化技术,从夏黑葡萄中筛选分离到5株附生细菌,提取菌株的基因组DNA,PCR扩增16S r DNA序列,产物纯化后进行克隆测序,利用MEGA 6.0软件对序列进行同源性比对分析,构建系统进化树后,将5株附生细菌分别鉴定为克雷伯肺炎杆菌(Klebsiella pneumoniae)、枯草芽孢杆菌(Bacillus subtilis)、克考氏菌(Kocuria marina)、嗜气芽孢杆菌(Bacillus aerophilus)、类芽孢杆菌(Paenibacillus agaridevorans)。在此基础上,以非核糖体多肽合成酶基因(non-ribosomal peptide compounds synthetase,NRPS)为靶点,筛选到含有目的条带的菌株PTWA2,经系统发育树预测该菌株能够产生环脂肽类化合物。该菌株发酵产物经有机溶剂萃取、常压硅胶柱层析、Sephedex LH-20等分离手段纯化得到一个单体化合物,采用核磁共振和质谱方法鉴定该化合物为环脂肽Surfactin A。该研究结果为以功能基因定向筛选产生环脂肽化合物的菌株提供了新的研究方法与理论依据。
[Abstract]:In this study, 5 epiphytic bacteria were isolated from summer black grape by plate dilution method and oblique surface purification technique. The 16s r DNA sequence was amplified by genomic DNA,PCR of the isolated strain, and cloned and sequenced after purification. The sequence was analyzed by MEGA 6.0, and phylogenetic tree was constructed. Five strains of epiphytic bacteria were identified as Klebsiella pneumoniae (Klebsiella pneumoniae), Bacillus subtilis (Bacillus subtilis), Kirschner's bacillus (Kocuria marina), respectively. Bacillus aerophilus (Bacillus aerophilus), Bacillus class (Paenibacillus agaridevorans). On this basis, the non-ribosomal polypeptide synthase gene (non-ribosomal peptide compounds synthetase,NRPS) was used as the target, and the strain PTWA2, containing the target band was selected. The phylogenetic tree was used to predict that the strain could produce cyclic peptide compounds. The fermentation product of the strain was extracted by organic solvent, separated by silica gel column chromatography under atmospheric pressure, and purified by Sephedex LH-20. The compound was identified as a cyclic peptide Surfactin A by NMR and MS. The results provide a new method and theoretical basis for the screening of cyclopeptide producing strains by functional gene orientation.
【作者单位】： 南京农业大学食品科技学院;
【基金】：2016年农产品质量安全风险评估项目(GJFP201601205)
【分类号】：TS255.1
本文编号：2306633
[Abstract]:In this study, 5 epiphytic bacteria were isolated from summer black grape by plate dilution method and oblique surface purification technique. The 16s r DNA sequence was amplified by genomic DNA,PCR of the isolated strain, and cloned and sequenced after purification. The sequence was analyzed by MEGA 6.0, and phylogenetic tree was constructed. Five strains of epiphytic bacteria were identified as Klebsiella pneumoniae (Klebsiella pneumoniae), Bacillus subtilis (Bacillus subtilis), Kirschner's bacillus (Kocuria marina), respectively. Bacillus aerophilus (Bacillus aerophilus), Bacillus class (Paenibacillus agaridevorans). On this basis, the non-ribosomal polypeptide synthase gene (non-ribosomal peptide compounds synthetase,NRPS) was used as the target, and the strain PTWA2, containing the target band was selected. The phylogenetic tree was used to predict that the strain could produce cyclic peptide compounds. The fermentation product of the strain was extracted by organic solvent, separated by silica gel column chromatography under atmospheric pressure, and purified by Sephedex LH-20. The compound was identified as a cyclic peptide Surfactin A by NMR and MS. The results provide a new method and theoretical basis for the screening of cyclopeptide producing strains by functional gene orientation.
【作者单位】： 南京农业大学食品科技学院;
【基金】：2016年农产品质量安全风险评估项目(GJFP201601205)
【分类号】：TS255.1
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