轮枝镰孢的荧光标记及其在寄主—病原菌互作中的应用
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摘要
玉米茎腐病在中国乃至全世界玉米生产区都有普遍发生。近年来,受耕作制度(如保护性耕作、秸秆还田等)的改变和气候变化的影响,我国玉米茎腐病的发生正逐渐加重。玉米茎腐病的主要致病菌之一轮枝镰孢[Fusarium verticillioides (Sacc.) Nirenberg]不仅能侵染玉米的根、茎和穗,引起苗枯病、茎腐病和穗腐病,还能像内生菌那样既能定殖于植株体内又不引起病症。
本研究采用农杆菌介导法将红色荧光蛋白基因DsRed转入轮枝镰孢FV菌株,通过对转化子的生理生化等分析筛选到与野生菌差异最小的转化子进行后续分析。主要结论如下:农杆菌介导法能够将GFP和DsRed荧光蛋白标记基因随机插入轮枝镰孢的基因组内,且外源基因的差异多为单拷贝随机插入。对转化子进行生理生化等指标的测定,发现DsRed更易导致突变体的出现。采用农杆菌介导法将红色荧光蛋白基因DsRed转入轮枝镰孢FV菌株,利用荧光显微镜观察轮枝镰孢在玉米感抗自交系根部定殖和生长的规律。
在感病材料中土壤中的轮枝镰孢首先从须根开始,它们可以在须根中大量定殖而不使须根致死。所以须根在轮枝镰孢侵染玉米以及玉米抗侵染方面可能起着关键作用,特别是在侵染的早期。随后轮枝镰孢沿主根向上侵染,以菌丝的形式扩展到地上组织。有些孢子附着在根表皮的纹理中,萌发形成菌丝而扩展;有的则向内侵染附着的细胞,然后再继续向周边侵染。由根内部向上侵染的菌丝多沿着细胞间隙上行,有些也会穿行在不同细胞之间。轮枝镰孢在侵染感病玉米有时会形成马赛克结构,而且这种马赛克结构在后期菌丝充满整个植物细胞时,会造成细胞的死亡。菌丝通常会定殖在须根下面或附近的细胞中。而在抗病玉米材料中,其须根数量明显低于感病材料,而且会形成坏死斑以限制轮枝镰孢的扩展。分析接种不同时间轮枝镰孢在玉米根和茎基部组织形成的单菌落数量(CFU)发现,轮枝镰孢在根部的CFU值随时间逐渐减小,而茎基部的CFU值则呈逐渐增大的趋势。这说明土壤中的轮枝镰孢能够通过对根系的系统侵染途径危害地上部组织。在抗病材料中根部和茎基部的CFU值都低于感病材料。
Maize stalk rot occures widely all over the world. In recent years, this disease was ingravescent in China due to the shift of farming system (e.g., protective farming and straw returning) and weather conditions. Fusarium verticillioides (Sacc.) Nirenberg, one of the major pathogens of stalk rot in maize (Zea mays L.), is able to infect maize root, stalk, and ear, inciting seedling blight, stalk rot, and ear rot. It also can grow inside maize tissues like asymptomatic endophyte.
In this study, gene DsRed encoding red fluorescent protein was delivered into F. verticillioides strain FV via Agrobacterium tumefaciens-mediated transformation (ATMT). the mutant, which showed the minimum differences with FV was selected by physio-biochemical characteristics analysis, was employed for further study. The main results as followed: fluorescent protein GFP and DsRed were inserted in the genome of F. verticillioidies by ATMT randomly. The results of physio-biochemical characteristics analysis showed the F. verticillioidies labelled with DsRed tended to form mutants. In this study, gene DsRed encoding red fluorescent protein was delivered into F. verticillioides strain FV via Agrobacterium tumefaciens-mediated transformation. Using the DsRed-tagged F. verticillioides, systemic colonization of the fungus in roots and stems of the susceptible and resistance maize inbred were investigated.
In susceptible maize lines,the fungus invaded and multiplied inside the hair root tissues at the very beginning, and the fungus were able to colonize in hair roots without killing them. Then the hyphae of Fusarium verticillioidies expanded upward along the main roots. Some conidia adhered to the veins of root surfaces and germinated to produce hyphae, which expanded upward. The hyphae were able to expand along the intercellular space or ran across several cells by breaking into them. Fusarium verticillioidies was able to form the mosaic pattens, when they was propagated in susceptable maize roots, and the cells seems to be killed when they fill with hyphea. The hyphea alway located under maize root surface with some hair roots grew on it. The resistent maize lines, which developed less hair roots than susceptable maize lines, usually form the necrotispot to limit the expension of Fusarium verticillioidies. Analysis of colony forming unit (CFU) from diseased plants demonstrated that following inoculation the CFU values from roots tended to decrease, but those from stems inclined to increase. This indicates that F. verticillioides is able to attack the aboveground parts of plant via systemic colonization on roots.
本研究采用农杆菌介导法将红色荧光蛋白基因DsRed转入轮枝镰孢FV菌株,通过对转化子的生理生化等分析筛选到与野生菌差异最小的转化子进行后续分析。主要结论如下:农杆菌介导法能够将GFP和DsRed荧光蛋白标记基因随机插入轮枝镰孢的基因组内,且外源基因的差异多为单拷贝随机插入。对转化子进行生理生化等指标的测定,发现DsRed更易导致突变体的出现。采用农杆菌介导法将红色荧光蛋白基因DsRed转入轮枝镰孢FV菌株,利用荧光显微镜观察轮枝镰孢在玉米感抗自交系根部定殖和生长的规律。
在感病材料中土壤中的轮枝镰孢首先从须根开始,它们可以在须根中大量定殖而不使须根致死。所以须根在轮枝镰孢侵染玉米以及玉米抗侵染方面可能起着关键作用,特别是在侵染的早期。随后轮枝镰孢沿主根向上侵染,以菌丝的形式扩展到地上组织。有些孢子附着在根表皮的纹理中,萌发形成菌丝而扩展;有的则向内侵染附着的细胞,然后再继续向周边侵染。由根内部向上侵染的菌丝多沿着细胞间隙上行,有些也会穿行在不同细胞之间。轮枝镰孢在侵染感病玉米有时会形成马赛克结构,而且这种马赛克结构在后期菌丝充满整个植物细胞时,会造成细胞的死亡。菌丝通常会定殖在须根下面或附近的细胞中。而在抗病玉米材料中,其须根数量明显低于感病材料,而且会形成坏死斑以限制轮枝镰孢的扩展。分析接种不同时间轮枝镰孢在玉米根和茎基部组织形成的单菌落数量(CFU)发现,轮枝镰孢在根部的CFU值随时间逐渐减小,而茎基部的CFU值则呈逐渐增大的趋势。这说明土壤中的轮枝镰孢能够通过对根系的系统侵染途径危害地上部组织。在抗病材料中根部和茎基部的CFU值都低于感病材料。
Maize stalk rot occures widely all over the world. In recent years, this disease was ingravescent in China due to the shift of farming system (e.g., protective farming and straw returning) and weather conditions. Fusarium verticillioides (Sacc.) Nirenberg, one of the major pathogens of stalk rot in maize (Zea mays L.), is able to infect maize root, stalk, and ear, inciting seedling blight, stalk rot, and ear rot. It also can grow inside maize tissues like asymptomatic endophyte.
In this study, gene DsRed encoding red fluorescent protein was delivered into F. verticillioides strain FV via Agrobacterium tumefaciens-mediated transformation (ATMT). the mutant, which showed the minimum differences with FV was selected by physio-biochemical characteristics analysis, was employed for further study. The main results as followed: fluorescent protein GFP and DsRed were inserted in the genome of F. verticillioidies by ATMT randomly. The results of physio-biochemical characteristics analysis showed the F. verticillioidies labelled with DsRed tended to form mutants. In this study, gene DsRed encoding red fluorescent protein was delivered into F. verticillioides strain FV via Agrobacterium tumefaciens-mediated transformation. Using the DsRed-tagged F. verticillioides, systemic colonization of the fungus in roots and stems of the susceptible and resistance maize inbred were investigated.
In susceptible maize lines,the fungus invaded and multiplied inside the hair root tissues at the very beginning, and the fungus were able to colonize in hair roots without killing them. Then the hyphae of Fusarium verticillioidies expanded upward along the main roots. Some conidia adhered to the veins of root surfaces and germinated to produce hyphae, which expanded upward. The hyphae were able to expand along the intercellular space or ran across several cells by breaking into them. Fusarium verticillioidies was able to form the mosaic pattens, when they was propagated in susceptable maize roots, and the cells seems to be killed when they fill with hyphea. The hyphea alway located under maize root surface with some hair roots grew on it. The resistent maize lines, which developed less hair roots than susceptable maize lines, usually form the necrotispot to limit the expension of Fusarium verticillioidies. Analysis of colony forming unit (CFU) from diseased plants demonstrated that following inoculation the CFU values from roots tended to decrease, but those from stems inclined to increase. This indicates that F. verticillioides is able to attack the aboveground parts of plant via systemic colonization on roots.
引文
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