桃褐腐病生防细菌FD6硝吡咯菌素合成基因簇的克隆及prnA功能分析

Cloning of Pyrrolnitrin Synthetic Gene Cluster prn and prn A Functional Analysis from Antagnistic Bacteria FD6 Against Peach Brown Rot

荧光假单胞菌(Pseudomonas fluorescens)FD6分离自福建闽侯青口青菜根围土壤,采用凹玻片法和离体果实接种法测定菌株FD6对桃褐腐病菌(Monilinia fructicola)的抑制能力。PCR法克隆硝吡咯菌素合成基因簇结构基因(prn),利用同源重组构建prn A缺失突变体并分析其功能。结果表明,菌株FD6处理桃后可完全抑制桃褐腐病发生;细菌悬浮液对褐腐病菌分生孢子的萌发抑制率达93.18%,细菌培养滤液的抑制率为69.36%;细菌悬浮液对黄瓜根结线虫也具有较强的致死作用。序列分析表明荧光假单胞菌FD6硝吡咯菌素合成基因簇全长为5 868 bp,内含4个开放阅读框prn A、prn B、prnC和prn D,这4个基因组成1个共转录单元。系统发育进化树显示菌株FD6与P.protegens CHA0、Pf-5的prn基因相似性达94%。利用遗传学方法证实prn A基因是硝吡咯菌素合成的必需因子,此外该基因还影响2,4–二乙酰基间苯三酚和藤黄绿脓菌素的合成。

Pseudomonas fluorescens FD6 was isolated from the canola rhizosphere in Fujian.To define the inhibit ability of the strain FD6 to Monilinia fructicola,the causal agent of peach brown rot was detected using microscope concave slides method and on detached peach fruits.The pyrrolnitrin biosynthetic gene cluster was cloned using PCR amplification,and a prnA-deletion mutant was generated by homologous recombinant to identify its function.The results showed that strain FD6 could control completely brown rot on peach fruits.The bacterial culture of strain FD6 inhibited the spores of M.fructicola germination significantly by 93.18%,whereas the cultural filtrate of the strain FD6 only showed a lower inhibition to M.fructicola by 69.36%.The bacterium culture showed that high nematicidal activity to Meloidogyne incognita.The obtained 5 868 bp prn contained four genes prnA,prnB,prnC and prnD organized in a single transcript unit.The phylogenetic analysis indicated that prn of strain FD6 identified with that of P.protegens CHAO and Pf-5 by 94%.prnA was required for PRN synthesis in P.fluorescens FD6,and involved in the biosynthesis of 2,4-diacetylphloroglucinol and pyoluteorin as well.