基于原生质体转化的甘蔗梢腐病菌YN41基因敲除体系的构建

Construction of gene knockout system in sugarcane pokkah boeng pathogen Fusarium proliferatum YN41 based on transformation of protoplast

基因敲除技术日益成为基因功能研究的重要手段,为了深入研究甘蔗梢腐病菌致病基因的功能,构建了梢腐病菌YN41菌株的基因敲除体系。本研究构建了基于线性DNA的同源重组基因敲除技术,融合PCR构建敲除盒即带有潮霉素基因标记的线性DNA融合片段,配置0.05 g·mL^(-1)溶壁酶+0.01 g·mL^(-1)崩溃酶的复合酶液28℃酶解3 h获得了高产量的原生质体,利用聚乙二醇(PEG)介导的原生质体的转化,PCR鉴定技术和酶切鉴定技术对转化子进行鉴定,荧光定量PCR分析和Southern Blot方法进一步对基因缺失突变体进行验证,生物学表型(菌落形态、生长速率、产孢量、生物量和致病力)验证了PK基因敲除体系的成功构建。28℃酶解3 h获得了2×10^(7)个·mL^(-1)原生质体;对编码丙酮酸激酶的PK基因进行了基因敲除验证,获得了纯合敲除突变体;荧光定量PCR检测转录水平无表达;Southern Blot结果显示使用PK基因探针杂交,基因缺失突变体无条带,野生型杂交到目的条带;生物学表型验证了PK基因敲除突变体对比野生型菌丝色素加深,生长速率先慢后快、产孢量增加,致病力增强,成功构建了PK基因敲除突变体。甘蔗梢腐病菌YN41基因敲除体系的成功构建,为甘蔗梢腐病菌致病基因的功能研究提供了技术平台。

Gene-knockout has been becoming an important tool to verify gene functions in vivo. In order to further study the function of pathogenic genes of sugarcane Pokkah boeng pathogen Fusarium proliferatum, a gene knockout system for YN41 strain was created. In this paper, we constructed a linear hph selective marker cassette flanking upstream and downstream homologous fragments, by fusion PCR;A mixture solution of 0.01 g·mL^(-1)driselase and 0.05 g·mL^(-1)yase was optimized to produce the most high yield of protoplast was 3 h at 28 ℃;Protoplast transformation was mediated by polyethylene glycol(PEG);The transformants were identified by PCR and enzyme digestion. The gene deletion mutant was further verified by fluorescence quantitative PCR and Southern Blot. The biological phenotype,(such as colony morphology, growth rate, sporulation, biomass and pathogenicity), indicated the successful construction of gene knockout system. 2×10^(7) protoplasts per milliliter were obtained by enzymatic hydrolysis at 28 ℃ for 3 h;The PK gene encoding pyruvate kinase was knocked out and homozygous was obtained. Real-time PCR detects no expression at the transcription level. Southern blot showed that there was no band in the gene deletion mutant using PK gene probe, but the wild type hybridized to the target band The PK gene knockout mutant exhibited an increased spre production, enhanced pathogenicity, and the varied growth rate which was slower firster and compared with wild type. The gene knockout system for F. proliferatum YN41 was successfully developed which provided a technical platform for the functional research on the pathogenic genes.