【摘要】：本研究采用平板稀釋法和斜面分離純化技術(shù),從夏黑葡萄中篩選分離到5株附生細(xì)菌,提取菌株的基因組DNA,PCR擴(kuò)增16S r DNA序列,產(chǎn)物純化后進(jìn)行克隆測(cè)序,利用MEGA 6.0軟件對(duì)序列進(jìn)行同源性比對(duì)分析,構(gòu)建系統(tǒng)進(jìn)化樹(shù)后,將5株附生細(xì)菌分別鑒定為克雷伯肺炎桿菌(Klebsiella pneumoniae)、枯草芽孢桿菌(Bacillus subtilis)、克考氏菌(Kocuria marina)、嗜氣芽孢桿菌(Bacillus aerophilus)、類芽孢桿菌(Paenibacillus agaridevorans)。在此基礎(chǔ)上,以非核糖體多肽合成酶基因(non-ribosomal peptide compounds synthetase,NRPS)為靶點(diǎn),篩選到含有目的條帶的菌株P(guān)TWA2,經(jīng)系統(tǒng)發(fā)育樹(shù)預(yù)測(cè)該菌株能夠產(chǎn)生環(huán)脂肽類化合物。該菌株發(fā)酵產(chǎn)物經(jīng)有機(jī)溶劑萃取、常壓硅膠柱層析、Sephedex LH-20等分離手段純化得到一個(gè)單體化合物,采用核磁共振和質(zhì)譜方法鑒定該化合物為環(huán)脂肽Surfactin A。該研究結(jié)果為以功能基因定向篩選產(chǎn)生環(huán)脂肽化合物的菌株提供了新的研究方法與理論依據(jù)。
[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.
【作者單位】： 南京農(nóng)業(yè)大學(xué)食品科技學(xué)院;
【基金】：2016年農(nóng)產(chǎn)品質(zhì)量安全風(fēng)險(xiǎn)評(píng)估項(xiàng)目(GJFP201601205)
【分類號(hào)】：TS255.1

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