Establishment of remi mutagenesis of setosphaeria turcica and characterization of mutants | 玉米大斑病菌REMI体系的建立及突变体分析

Chinese. 【目的】建立高效的玉米大斑病菌REMI遗传转化体系,进而利用该技术创制突变体,为克隆病菌生长发育和致病性相关的基因奠定基础。【方法】摸索玉米大斑病菌原生质体制备的菌龄、酶系统及酶解条件、原生质体收集方式、转化子最适潮霉素B筛选浓度以及转化使用的质粒,建立该病菌高效的REMI转化体系;利用PCR技术对潮霉素B抗性转化子进行筛选并对部分突变体进行分析。【结果】培养20 h的幼嫩菌丝,用浓度分别达到 1.25%的Lyallzyme、Snailase和Drislase 3种酶混和液酶解4 h、离心速度为3 000 r/min时原生质体产量最大;当潮霉素B浓度为50 μg?mL-1时,野生型菌株的分生孢子萌发和菌落生长完全受到抑制,可选用该浓度作为转化子的筛选浓度;使用pAN7-1转化效率较高,对利用该体系转化获得的大量转化子进行PCR检测中筛选出了部分产孢量和致病力变化的突变菌株。【结论】建立了玉米大斑病菌的REMI遗传转化体系,将为玉米大斑病菌突变体库的建立和致病相关基因的克隆奠定基础。

English. 【Objective】 The objective of this study is to establish high efficient REMI mutagenesis system, which is able to construct mutants that can be used to clone genes related to growth, development and pathogenicity of S. turcica.【Method】 REMI mutagenesis system was established through seeking the optimal different ages of this fungus, cell wall digest enzymes and its condition and collection method which affect protoplast yield, and seeking hygromycin B concentration for transformants screening, and seeking plasmid for transformation, which was used to achieve transformants. 【Result】 When lyallzyme, snailase, and drislase were mixed and at concentration of 1.25%, respectively, and when mycelium was cultured for 20 h, and 3 000 r/min for collection, the protoplast production was the maximum. Hygromycin B at 50 μg?mL-1 was determined as the optimal concentration for transformants screening, in which conidial germination and colony growth of wild type was completely inhibited. The plasmid pAN7-1 had higher transformation efficiency. The optimal system was used to induce mutagenesis and lots of transformants were obtained, from which some conidia production-changed and pathogencity-changed transformants were found. 【Conclusion】 The REMI mutagenesis of S. turcica was established, which had a great significance in construction of mutant library and in cloning the pathogencity-related genes.