高产吩嗪-1-甲酰胺的绿针假单胞菌的诱变与基因工程育种

Mutagenesis and Genetic Engineering Breed of Pseudomonas chlororaphis with High Yield of Phenaizne-1-Carboximade

产量低是限制生物农药吩嗪-1-甲酰胺(Phenaizne-1-carboximade,PCN)推广应用的主要因素。本文综合使用诱变育种和基因工程改造来提高绿针假单胞菌的PCN产量。诱变育种使用亚硝基胍(NTG)和紫外线(UV)处理绿针假单胞菌HT66野生型菌株,以平板菌落表面的绿色PCN晶体量为指标建立高通量诱变筛选方法,通过10轮稳定可靠的诱变处理,获得的诱变高产株P3中PCN产量达到1 697mg/L,是野生菌株的3.99倍。在固体平板上培养时,P3菌株的菌落更大,PCN晶体更多且出现更早。在诱变育种的基础上进一步进行基因工程改造,敲除P3株中负调控PCN合成的rpeA基因,获得的P3ΔrpeA菌株PCN产量达到2 167mg/L,充分证明了2种育种方法结合使用的高效性。

The low production is the main barrier for phenaizne-1-carboximade（PCN）to be applied to agricultural process.In this paper,mutation breeding and genetic engineering breeding were combined to obtain the high yield PCN from Pseudomonaschlororaphisstrain HT66.In the mutation breed,strain P3 was obtained by crossing mutagenesis of the NTG and UV treatment from wild-strain.The highthroughput screening method was established by the amount of PCN crystals on the surface of colony.PCN production of strain P3 was 1697mg/L,which was 3.99 times more than strain HT66;The colony of the strain P3 was bigger with more and earlier PCN crystals on the surface.Genetic engineering was carried out on the basis of mutation breeding.The negative regulator to PCN biosyntheisis,rpeAin the strain P3 was eliminated.The PCN production of strain P3ΔrpeA was as much as 2 167 mg/L.It was proved that the combination of two breeding methods is more efficient.