原生质体融合选育高产脂肽LI-F多粘类芽胞杆菌及融合菌株表达差异分析

Breeding of high-producing LI-F lipopeptide Paenibacillus polymyxa by protoplast fusion and differential expression analysis of fusion strains

摘要:

本研究以多粘类芽胞杆菌JSa-9前期诱变获得的两株带有营养缺陷型标记的菌株N1-37(Phe–)和N2-27(His–)作为亲本菌株，采用聚乙二醇作为促融剂，进行原生质体融合，筛选出高产LI-F类抗菌脂肽的融合菌株。通过HPLC对融合菌株和原始菌株产LI-F类抗菌脂肽进行定量检测，并利用实时荧光定量PCR对菌株JSa-9和融合菌株中LI-F类抗菌脂肽合成酶的关键基因fusA1、fusA2、fusC1和fusC2的差异性表达进行分析。结果表明，经过原生质融合获得一株LI-F类抗菌脂肽高产菌株F5-15。菌株F5-15的LI-F类抗菌脂肽产量为原始菌株产量的3.1倍，LI-F类抗菌脂肽合成酶的4个关键基因在融合菌株F5-15中的表达量分别是其在原始菌株JSa-9中的10.48、2.48、2.1和11.8倍。

Abstract:

Auxotrophic strains of N1-37 (Phe–) and N2-27 (His–), screened from mutations of Paenibacillus polymyxa JSa-9 previously, were used as the parent strains to screen high-producing LI-F antibacterial lipopeptide fusion strain through protoplast fusion with polyethylene glycol as a promote agent. Fusion strain F5-15 was obtained. Then the product of LI-F antibacterial lipopeptide was quantified by HPLC, and the difference of expression of the key genes of lipopeptide synthase between wild strain JSa-9 and the fusion strain was analyzed by real-time PCR. LI-F antibacterial lipopeptide yield of the fusion strain F5-15 was 3.1-fold of the original strain JSa9’s, and the expression levels of the target genes were 10.48, 2.48, 2.1 and 11.8 fold of?the initial strain JSa-9, respectively.