拟轮枝镰孢ATMT突变体库的构建及分析
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摘要
【目的】通过建立适用于拟轮枝镰孢(Fusarium verticillioides)的农杆菌介导遗传转化体系,构建绿色荧光蛋白(green fluorescent protein,GFP)标记的拟轮枝镰孢ATMT(Agrobacterium tumefaciens-mediated transformation)突变体库,并对突变体库进行筛选分析,为研究拟轮枝镰孢在玉米果穗上的侵染途径和致病的分子机制打下基础。【方法】筛选头孢噻肟钠(cefotaxime sodium,Cefo)和氨苄青霉素钠(ampicillin sodium,Amp)对根癌农杆菌AGL-1的抑菌浓度和拟轮枝镰孢对潮霉素B(hygromycin B)的敏感浓度;以含有绿色荧光蛋白基因(GFP)、潮霉素抗性基因(HPH)的穿梭质粒为载体,通过ATMT构建GFP标记的拟轮枝镰孢突变体库;利用潮霉素抗性筛选、GFP的特异性引物进行PCR检测和荧光显微镜观察,检测分析T-DNA插入情况及转化子稳定性;从突变体库中随机挑选9个转化子菌株并进行分析,对其产孢量、分生孢子萌发率、致病力等进行测定。【结果】通过农杆菌抑菌试验发现当Cefo/Amp的浓度为150/150μg·mL~(-1)时,AGL-1生长受到抑制;当潮霉素B的浓度为150μg·mL~(-1)时,拟轮枝镰孢完全丧失生长能力。利用优化后的ATMT转化获得了2 465株GFP标记的拟轮枝镰孢转化子;转化子在不含潮霉素B的PDA培养基上连续转接5代再转到含潮霉素B的培养基上仍能正常生长,说明HPH成功插入野生型基因组且稳定遗传;利用GFP特异性引物对转化子进行PCR检测,测序结果显示与NCBI中GFP(登录号:LC420351.1)的同源性为99.26%,表明GFP已成功整合到野生型基因组中;转化子菌丝和孢子在荧光显微镜下观察均呈现绿色,而野生型菌株未观察到任何荧光,表明GFP转移到拟轮枝镰孢野生型菌株基因组中,且能够成功表达。对部分转化子分析发现,与野生型相比转化子54的产孢量明显增多,约为野生型的1.9倍;转化子24的分生孢子萌发率在相同时间内明显下降;转化子13的致病力增强,病害级别达到9级,转化子33和16致病力减弱为3级,转化子4致病力最弱为1级,部分转化子生物学性状未发生明显变化。【结论】构建了农杆菌介导GFP标记的拟轮枝镰孢突变体库,筛选分析获得了产孢量、孢子萌发率、致病力发生变化的突变体,为进一步研究拟轮枝镰孢侵染玉米果穗的途径和致病的分子机制打下了基础。
【Objective】The objective of this study is to establish a highly efficient ATMT mutagenesis system of Fusarium verticillioides, and to construct ATMT mutant library in which mutant contains green fluorescent protein(GFP). And then this library was used for screening and analysis, which can lay a foundation for studying the infection pathway and molecular pathogenesis of F.verticillioides on maize ear. 【Method】 The inhibitory concentration of cefotaxime sodium(Cefo) and ampicillin sodium(Amp)against Agrobacterium tumefaciens AGL-1 and the sensitive concentration of hygromycin B against F. verticillioides were screened for ATMT mutagenesis system. A shuttle plasmid containing GFP and hygromycin phosphotransferase(HPH) genes was used as a vector to construct the ATMT mutants library of F. verticillioides. The T-DNA insertion and stability of transformant were detected and analyzed through hygromycin B resistance, PCR identification of GFP-specific primers, and fluorescence microscopy. Nine transformants were randomly selected and the sporulation number, conidial germination rate, and pathogenicity were measured.【Result】When the concentration of Cefo/Amp was 150/150 μg·mL~(-1), the growth of AGL-1 was inhibited, and when the concentration of hygromycin B was 150 μg·mL~(-1), the growth of F. verticillioides was completely incapacitated. Using the optimized ATMT, a total of 2 465 GFP-labeled transformants were obtained, these transformants could still grow normally on PDA medium containing hygromycin B after cultured 5 generations on hygromycin-free PDA medium, which indicated that HPH had been integrated into wild-type(WT) genome of F. verticillioides and the transformants were stable in their characteristics of genetics. The PCR detection results amplified with GFP-specific primers showed that the homology of transformants with GFP(accession number:LC420351.1) in NCBI was 99.26%, and the hyphae and conidia of transformants showed green fluorescence under the fluorescence microscope, while no fluorescence was observed in WT, indicating that GFP had been integrated into the WT genome and successfully expressed. Compared with WT strain, the sporulation number of transformant 54 increased significantly, about 1.9 times of that of WT strain, and the conidial germination rate of transformant 24 decreased obviously in the same time. The pathogenicity of transformant 13 was enhanced, the disease grade reached 9, the pathogenicity of transformant 33 and 16 was reduced to grade 3, and the pathogenicity of transformant 4 was the weakest, the disease grade was 1. There was no significant change in biological characters of partial transformants.【Conclusion】ATMT mutant library with GFP of F. verticillioides was constructed, and the mutants with changed sporulation number, conidia germination rate and pathogenicity were obtained through primarily screening. It will lay a foundation for further study on the infection pathway and pathogenic molecular mechanisms of F. verticillioides on maize ear in the future.
【Objective】The objective of this study is to establish a highly efficient ATMT mutagenesis system of Fusarium verticillioides, and to construct ATMT mutant library in which mutant contains green fluorescent protein(GFP). And then this library was used for screening and analysis, which can lay a foundation for studying the infection pathway and molecular pathogenesis of F.verticillioides on maize ear. 【Method】 The inhibitory concentration of cefotaxime sodium(Cefo) and ampicillin sodium(Amp)against Agrobacterium tumefaciens AGL-1 and the sensitive concentration of hygromycin B against F. verticillioides were screened for ATMT mutagenesis system. A shuttle plasmid containing GFP and hygromycin phosphotransferase(HPH) genes was used as a vector to construct the ATMT mutants library of F. verticillioides. The T-DNA insertion and stability of transformant were detected and analyzed through hygromycin B resistance, PCR identification of GFP-specific primers, and fluorescence microscopy. Nine transformants were randomly selected and the sporulation number, conidial germination rate, and pathogenicity were measured.【Result】When the concentration of Cefo/Amp was 150/150 μg·mL~(-1), the growth of AGL-1 was inhibited, and when the concentration of hygromycin B was 150 μg·mL~(-1), the growth of F. verticillioides was completely incapacitated. Using the optimized ATMT, a total of 2 465 GFP-labeled transformants were obtained, these transformants could still grow normally on PDA medium containing hygromycin B after cultured 5 generations on hygromycin-free PDA medium, which indicated that HPH had been integrated into wild-type(WT) genome of F. verticillioides and the transformants were stable in their characteristics of genetics. The PCR detection results amplified with GFP-specific primers showed that the homology of transformants with GFP(accession number:LC420351.1) in NCBI was 99.26%, and the hyphae and conidia of transformants showed green fluorescence under the fluorescence microscope, while no fluorescence was observed in WT, indicating that GFP had been integrated into the WT genome and successfully expressed. Compared with WT strain, the sporulation number of transformant 54 increased significantly, about 1.9 times of that of WT strain, and the conidial germination rate of transformant 24 decreased obviously in the same time. The pathogenicity of transformant 13 was enhanced, the disease grade reached 9, the pathogenicity of transformant 33 and 16 was reduced to grade 3, and the pathogenicity of transformant 4 was the weakest, the disease grade was 1. There was no significant change in biological characters of partial transformants.【Conclusion】ATMT mutant library with GFP of F. verticillioides was constructed, and the mutants with changed sporulation number, conidia germination rate and pathogenicity were obtained through primarily screening. It will lay a foundation for further study on the infection pathway and pathogenic molecular mechanisms of F. verticillioides on maize ear in the future.
引文
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[3]DUAN C X,QIN Z H,YANG Z H,LI W X,SUN S L,ZHU Z D,WANG X M.Identification of pathogenic Fusarium spp.causing maize ear rot and potential mycotoxin production in China.Toxins,2016,8(6):186.
[4]LANUBILE A,BERNARDI J,MAROCCO A,LOGRIECO A,PACIOLLA C.Differential activation of defense genes and enzymes in maize genotypes with contrasting levels of resistance to Fusarium verticillioides.Environmental and Experimental Botany,2012,78:39-46.
[5]ZHOU D N,WANG X M,CHEN G K,SUN S L,YANG Y,ZHU Z D,DUAN C X.The major Fusarium species causing maize ear and kernel rot and their toxigenicity in Chongqing,China.Toxins,2018,10(2):90.
[6]PROCTOR R H,BROWN D W,PLATTNER R D,DESJARDINS AE.Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis.Fungal Genetics and Biology,2003,38(2):237-249.
[7]KIM H,WOLOSHUK C P.Role of AREA,a regulator of nitrogen metabolism,during colonization of maize kernels and fumonisin biosynthesis in Fusarium verticillioides.Fungal Genetics and Biology,2008,45(6):947-953.
[8]MYUNG K,ZITOMER N C,DUVALL M,GLENN A E,RILEY R T,CALVO A M.The conserved global regulator VeA is necessary for symptom production and mycotoxin synthesis in maize seedlings by Fusarium verticillioides.Plant Pathology,2012,61(1):152-160.
[9]BLUHM B H,KIM H,BUTCHKO R A E,WOLOSHUK C P.Involvement of ZFR1 of Fusarium verticillioides in kernel colonization and the regulation of FST1,a putative sugar transporter gene required for fumonisin biosynthesis on maize kernels.Molecular Plant Pathology,2008,9(2):203-211.
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