马铃薯炭疽病菌的侵染过程及病原菌诱导基因差异表达分析
发布时间：2018-08-15 11:30
【摘要】：近年来,随着马铃薯种植面积的不断增加,马铃薯炭疽病(Potato Black Dot)越来越严重,引起马铃薯植株的早死和贮藏期薯块的大量腐烂,已成为马铃薯的主要病害之一。本研究采用PEG诱导原生质体转化的方法,借助ITS序列用GFP基因标记马铃薯炭疽病菌后观察其侵染过程,研究马铃薯在炭疽病菌胁迫下基因的差异表达情况,取得以下结论:1、马铃薯炭疽病菌的绿色荧光标记利用定点突变改造马铃薯炭疽病菌ITS序列后,构建了两侧含有ITS序列的GFP标记载体pITGH,通过同源重组法将hyg B::gfp基因标记到球炭疽菌的ITS序列上,获得稳定表达的转化子C-106、C-163和C-168,提取转化子的基因组DNA,经PCR扩增后,电泳检测到400bp的gfp基因预期片段,经测序表明gfp基因已整合至球炭疽菌基因组DNA中。2、转化子的生物学特性采用常规方法测定转化子的生物学特性,发现绿色荧光蛋白gfp基因的转化对球炭疽菌的菌落形态、生长速度和致病性等无影响,pH值对转化子的菌丝生长无影响,转化子在pH值为4~12的PDA培养基上均可生长,最适pH值为8。3、球炭疽菌对马铃薯的侵染过程将荧光标记菌株C-168接种于马铃薯茎秆和块茎,分别取样于荧光显微镜下观察其侵染马铃薯的过程,发现球炭疽菌菌丝在寄主茎秆组织中沿着细胞壁向相邻细胞进行延伸,且在维管束细胞中延伸速度最快,其次为周皮细胞和表皮细胞,在髓部细胞中最慢;在马铃薯块茎细胞中,菌丝沿着细胞壁向四周相邻细胞延伸,随着侵染时间的延长,菌丝交织呈网状裂解细胞结构,不断沿着细胞间隙向周围细胞延伸。4、马铃薯在球炭疽菌胁迫下的基因表达谱分析将球炭疽菌接种于马铃薯后,采用RNA-Seq技术进行测序,发现共有2936个基因发生差异表达,其中上调基因1 492和下调基因1 444。差异表达基因经过注释后GO功能分为3类,在生物学过程(biological process)中主要有刺激反应、应激反应和碳水化合物代谢过程等;在分子功能(Molecular Function)中主要涉及水解酶活性-作用于糖基键,裂解酶活性和四砒咯结合等;在细胞组成(cellular component)中主要有细胞外围、外区和外部封装结构等。差异表达基因显著性富集于24条Pathway,主要通路包括代谢途径、苯丙烷代谢途径、次生代谢产物的生物合成途径、光合作用-天线蛋白及淀粉和蔗糖代谢途径等。分析获得19个连续性共表达差异基因参与显著性富集代谢途径,包括次生代谢产物的生物合成、苯丙烷代谢途径和淀粉和蔗糖代谢途径等,均表现为下调表达。植物与病原物互作途径为非显著性富集代谢途径,涉及的差异表达基因有158个,其中有5个为连续性共表达的差异基因,1个上调表达,2个下调表达,2个先下调再上调表达。
[Abstract]:In recent years, with the increasing of potato planting area, the (Potato Black Dot) of potato anthracnose is becoming more and more serious, which causes the early death of potato plant and the mass rot of potato during storage, which has become one of the main diseases of potato. In this study, PEG was used to induce protoplast transformation, ITS sequence was used to mark potato anthracnose with GFP gene, and the infection process was observed, and the differential expression of potato genes under anthracnose stress was studied. The following conclusions were obtained: 1. The ITS sequence of potato anthracnose was modified by spot mutation with green fluorescent marker. A GFP marker vector pITGHcontaining ITS sequence was constructed. The hyg B::gfp gene was labeled into the ITS sequence of Bacillus anthracis by homologous recombination method. The stable expression transformants C-106, C-163 and C-168 were obtained. The genomic DNA of the transformant was extracted and amplified by PCR. The expected fragment of gfp gene of 400bp was detected by electrophoresis. Sequencing showed that the gfp gene had been integrated into the genomic DNA of Bacillus anthracis. The biological characteristics of the transformants were determined by conventional methods. It was found that the transformation of green fluorescent protein gfp gene had no effect on colony morphology, growth rate and pathogenicity of Bacillus anthracis. The pH value had no effect on the mycelial growth of the transformants, and the transformants could all grow on PDA medium with pH of 4 ~ 12. The optimum pH value was 8.3. The fluorescent labeled strain C-168 was inoculated into the stem and tuber of potato during the infection process of Bacillus anthracis, and the process of potato infection was observed under fluorescence microscope. It was found that the mycelium of Bacillus anthracis extended to the adjacent cells along the cell wall in the host stem, and the fastest extension was found in the vascular bundle cells, followed by pericarp cells and epidermal cells, and the slowest in the medullary cells. In potato tuber cells, the mycelium extends to adjacent cells along the cell wall. With the extension of infection time, the mycelium interweaves into a reticular cleavage cell structure. The gene expression profiles of potato under the stress of Bacillus anthracis were analyzed and sequenced by RNA-Seq technique. A total of 2936 genes were differentially expressed. Among them, the up-regulated gene 1492 and down-regulated gene 1444. The differentially expressed genes can be classified into three categories after being annotated. In the biological process of (biological process), they are mainly involved in stimulation response, stress response and carbohydrate metabolism, and in the molecular function (Molecular Function), the activity of hydrolase-acting on the glycosyl bond is mainly involved. The activity of lyase and the binding of tetraarsenicolus and so on, and the outer, outer region and outer encapsulated structure of (cellular component) were mainly found in the cell composition. The differentially expressed genes were significantly enriched in 24 pathways, including metabolic pathway, phenylpropane pathway, biosynthesis pathway of secondary metabolites, photosynthesis-antenna protein, starch and sucrose metabolism pathway. Nineteen continuous coexpression differentially expressed genes were found to be involved in significantly enriched metabolic pathways, including biosynthesis of secondary metabolites, phenylpropane metabolism pathway and starch and sucrose metabolic pathways, all of which showed down-regulated expression. There were 158 differentially expressed genes involved in the interaction between plant and pathogen. Among them, 5 genes were differentially expressed, 1 was up-regulated, 2 were down-regulated, and 2 were down-regulated and then upregulated.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.32
[Abstract]:In recent years, with the increasing of potato planting area, the (Potato Black Dot) of potato anthracnose is becoming more and more serious, which causes the early death of potato plant and the mass rot of potato during storage, which has become one of the main diseases of potato. In this study, PEG was used to induce protoplast transformation, ITS sequence was used to mark potato anthracnose with GFP gene, and the infection process was observed, and the differential expression of potato genes under anthracnose stress was studied. The following conclusions were obtained: 1. The ITS sequence of potato anthracnose was modified by spot mutation with green fluorescent marker. A GFP marker vector pITGHcontaining ITS sequence was constructed. The hyg B::gfp gene was labeled into the ITS sequence of Bacillus anthracis by homologous recombination method. The stable expression transformants C-106, C-163 and C-168 were obtained. The genomic DNA of the transformant was extracted and amplified by PCR. The expected fragment of gfp gene of 400bp was detected by electrophoresis. Sequencing showed that the gfp gene had been integrated into the genomic DNA of Bacillus anthracis. The biological characteristics of the transformants were determined by conventional methods. It was found that the transformation of green fluorescent protein gfp gene had no effect on colony morphology, growth rate and pathogenicity of Bacillus anthracis. The pH value had no effect on the mycelial growth of the transformants, and the transformants could all grow on PDA medium with pH of 4 ~ 12. The optimum pH value was 8.3. The fluorescent labeled strain C-168 was inoculated into the stem and tuber of potato during the infection process of Bacillus anthracis, and the process of potato infection was observed under fluorescence microscope. It was found that the mycelium of Bacillus anthracis extended to the adjacent cells along the cell wall in the host stem, and the fastest extension was found in the vascular bundle cells, followed by pericarp cells and epidermal cells, and the slowest in the medullary cells. In potato tuber cells, the mycelium extends to adjacent cells along the cell wall. With the extension of infection time, the mycelium interweaves into a reticular cleavage cell structure. The gene expression profiles of potato under the stress of Bacillus anthracis were analyzed and sequenced by RNA-Seq technique. A total of 2936 genes were differentially expressed. Among them, the up-regulated gene 1492 and down-regulated gene 1444. The differentially expressed genes can be classified into three categories after being annotated. In the biological process of (biological process), they are mainly involved in stimulation response, stress response and carbohydrate metabolism, and in the molecular function (Molecular Function), the activity of hydrolase-acting on the glycosyl bond is mainly involved. The activity of lyase and the binding of tetraarsenicolus and so on, and the outer, outer region and outer encapsulated structure of (cellular component) were mainly found in the cell composition. The differentially expressed genes were significantly enriched in 24 pathways, including metabolic pathway, phenylpropane pathway, biosynthesis pathway of secondary metabolites, photosynthesis-antenna protein, starch and sucrose metabolism pathway. Nineteen continuous coexpression differentially expressed genes were found to be involved in significantly enriched metabolic pathways, including biosynthesis of secondary metabolites, phenylpropane metabolism pathway and starch and sucrose metabolic pathways, all of which showed down-regulated expression. There were 158 differentially expressed genes involved in the interaction between plant and pathogen. Among them, 5 genes were differentially expressed, 1 was up-regulated, 2 were down-regulated, and 2 were down-regulated and then upregulated.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.32
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