摘要
NCD-2菌株是从棉花根际分离筛选到的一株枯草芽孢杆菌(Bacillus subtilis),对其作用机理研究发现该菌株不仅能够通过分泌抑菌物质对棉花黄萎菌等植物病原真菌起到抑制作用,同时还具有降解有机磷的能力。
通过电击转化将含有转座子mini-Tn10的质粒pHV1249转入NCD-2菌株中,并构建了NCD-2菌株的突变子库。从3000多个突变子中筛选到3株丧失解磷能力的突变子,Southern杂交实验证明突变株中转座子均为单拷贝插入,并且具有相同大小的杂交片断,因此认为这三个突变子可能具有相同的遗传背景。对其中一个突变子M216进行了以下研究。运用巢氏PCR技术对突变株M216中转座子插入位点基因的侧翼序列进行克隆和序列分析,结果表明丧失解磷能力突变株中转座子插入基因与B. subtilis 168菌株中phoR基因的相似率达到98%。将phoR基因克隆到pHY300PLK质粒上构建重组载体电击转化M216进行功能互补验证,互补菌株部分恢复了解磷能力,以上结果表明NCD-2菌株中phoR基因与其降解卵磷脂能力具有相关性。
本试验同时对NCD-2菌株产生的抑菌物质进行了分离和鉴定。利用盐酸沉淀、甲醇溶解的方法提取NCD-2菌株的脂肽类抗生素。双层培养基法测定提取物对棉花黄萎病菌、番茄灰霉病菌具有拮抗活性,进一步研究表明,抑菌物质能够影响真菌菌丝生长及孢子萌发。脂肽提取物经HPLC分析,获得3组主要的物质峰,其中组分Ⅱ具有抑菌活性。采用MALDI-TOF MS对组分Ⅱ进行质谱分析,确定组分Ⅱ中含有两组分子量相差14Da(CH2)的代谢同系物,一组物质的分子量依次为1446.801,1460.813,1474.830,1488.852,与脂肽类抗生素C15-C18Fengycin A对应分子量相差2Da,推测为Fengycin A的一种异构体,其脂肪酸碳链上具有一个双键；另一组同系物的分子量依次为1462.798,1476.817,1490.839,1504.855,1518.867,鉴定为脂肽类抗生素C14-C18 Fengycin B。
Bacillus subtilis strain NCD-2 was isolated from the rhizosphere of cotton, and showed antagonism to Verticillium dahliae by secreting antifungal active compounds. This strain also showed lecithin-solubilizing ability.
Plasmid pHV1249 which carrying transposon mini-Tn10 was transferred into strain NCD-2 by electrotransformation, and the transposon library of NCD-2 was constructed. Three mutants with defective of lecithin-solubilizing ability were selected from more than 3000 mini-Tn10 inserted mutants. Southern hybridization with mini-Tn10 fragment as a probe showed that all these mutants contained single copy transposon insertion, which possibly localized at the same chromosomal location. One mutant M216 was used for further study. The flanking sequence of insertion site was cloned from mutant M216 by Nest PCR. Sequence analysis showed that the inserted gene has of 98% identity with phoR in B. subtilis strain 168. Genetic complementation was conducted by cloning phoR gene into plasmid pHY300PLK and the recombinant plasmid was transferred into mutant M216. The lecithin-solubilizing ability of mutant M216 was restored. Results suggested that the lecithin-solubilizing ability of NCD-2 was related with phoR gene.
Purification and identification of antifungal lipopeptides produced by B. subtilis NCD-2 was also studied in this experiment. Crude lipopeptides were extracted with methanol from the precipitate, which was obtained by adding 6mol/L HC1 to the cell-free culture broth and then stored at 4℃overnight. The crude extract showed antifungal activities against V. dahliae and Botrytis cinerea on the double layer medium. Three mainly peak groups were obtained by HPLC analysis, and only the second compound had inhibitory activity against V. dahliae and B. cinerea. The second compound was analysised by MALDI-TOF MS and two groups of homologues with a difference of 14 Da in molecular weights were identified. In the first group, there were 4 kinds of homologues with the molecular weights of 1446.801,1460.813, 1474.830,1488.852Da, respectively, which were estimated as the isomer of Fengycin A(C15-18). In the second group, the molecular weights of 5 kinds of homologues were 1462.798,1476.817,1490.839,1504.855,1518.867Da, respectively, which were identified as lipopeptide antibiotics Fengycin B(C14-18).
引文
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