基于PEG介导原生质体转化构建粉红聚端孢荧光标记

Construction of Trichothecium roseum GFP tagged strains based on PEG-mediated protoplast transformation

粉红聚端孢是多种植物的重要病原菌。本研究通过酶解粉红聚端孢幼嫩菌丝细胞壁获得原生质体,用PEG介导原生质体转化将携带GFP基因和博来霉素抗性基因的外源DNA随机插入粉红聚端孢基因组,共获得博来霉素抗性菌株90株,转化效率达18个/μg。选取22株转化子荧光观察,发现均可表达荧光,菌株间荧光强度不同,其中10株转化子荧光表达较强。与野生型相比,突变菌株TR45的菌落生长、产孢量和致病力等生物学特性均未改变,在不含博来霉素的培养皿中继代培养10代荧光仍能稳定表达。本研究构建的高效原生质体制备和PEG介导粉红聚端孢遗传转化方法,可用于该菌基因功能研究,绿色荧光标记菌株可用于病菌侵入、田间监测、侵染循环等发生规律研究。

Trichothecium roseum is an important pathogen of various crops. Protoplasts of T. roseum were obtained via digesting cell walls of young hyphae by use of enzymatic solutions, and exogenous plasmid DNA carrying GFP gene and zeocin resistant gene was inserted into T. roseum genome by PEG mediated protoplast transformation. Totally 90 transformants were obtained from zeocin resistant screening plates, and DNA transformation efficiency reached 18 protoplasts/μg. Twenty-two transformants were used for observing fluorescence signals. Transformants showed green fluorescent signals on various fluorescent intensities depended on transformant individuals, and ten of them displayed relatively stronger fluorescent signals. There was no difference of biological characteristics including colonial morphology, conidiation as well as pathogenicity between the representative transformant TR45 and the wild type strain. As for transformant TR45 the green fluorescence was still stable after subculturing ten generations on plates without zeocin stress.The protocols of efficient protoplast preparation and PEG mediated protoplast transformation used in this study could be applied to explore functional genes of T. roseum. The obtained GFP tagged strains could be used for further researches on pathogen penetration, field detection and infection cycles of related diseases caused by T. roseum.