基于ARTP诱变和高通量筛选的绿针假单胞菌GP72育种方法

Breeding of Pseudomonas chlororaphis GP72 based on ARTP mutagenesis and high throughput screening

摘要:

【目的】绿针假单胞菌GP72是一种植物根围促生细菌，其分泌的次级代谢产物2-羟基-吩嗪(2-OH-PHZ)具有广谱抗真菌活性，但其产量较低，不能满足农业生产的应用需求，因此需对GP72进行改造，从而提高产量。【方法】从GP72的野生株出发，首次将2-OH-PHZ合成途径的限制性因子PhzO用绿色荧光蛋白(GFP)替换，以一种新型的常压室温等离子体技术(Atmospheric and room temperature plasma，ARTP)进行诱变，通过酶标仪测定96孔板中突变株的荧光强度进行高通量筛选；最后将荧光强度高的菌株中绿色荧光蛋白(Green fluorescent protein，GFP)替换为PhzO以获得2-OH-PHZ高产突变株。【结果】经过五轮诱变后，获得一株荧光强度增加1.62倍的突变株，用phzO基因回替后，该突变株在KB培养基中摇瓶培养时2-OH-PHZ的产量为野生型的4.62倍。【结论】基于安全、高效ARTP诱变技术，并以GFP替换限制性因子作为标记进行高通量筛选，可以快速获得高产2-OH-PHZ的GP72突变株，克服了传统诱变育种方法筛选难度大、费时费力的不足，为其它微生物的育种提供了参考。

Abstract:

[Objective] Pseudomonas chlororaphis GP72 is a plant growth-promoting rhizobacterium. Its secondary metabolite 2-hydroxy-phenazine (2-OH-PHZ) has broad-spectrum antimicrobial activity, although its yield is too low to apply in agricultural pest management. [Methods] The limiting factor PhzO in 2-OH-PHZ biosynthesis pathway was replaced by green fluorescent protein (GFP), and a novel atmospheric and room temperature plasma (ARTP) technology was used to mutagenize the strain. High-throughput screening of the mutants in a 96-well plate was done by using a microplate reader. Finally, GFP was replaced reversibly by PhzO in the strain with high fluorescence intensity to obtain the 2-OH-PHZ high-yield mutant. [Results] The yield of 2-OH-PHZ was 4.62 times higher than that of the wild-type strain in KB medium in shake flask culture. [Conclusion] The high yield of 2-hydroxy-phenazine GP72 mutants could be obtained rapidly and safely by high-throughput screening based on ARTP mutagenesis using GFP substitution of the restriction factor as the marker. This method overcomes the shortcomings of traditional breeding methods. This method could be used as a reference for other microbial breeding.