【摘要】：近年來(lái),隨著馬鈴薯種植面積的不斷增加,馬鈴薯炭疽病(Potato Black Dot)越來(lái)越嚴(yán)重,引起馬鈴薯植株的早死和貯藏期薯塊的大量腐爛,已成為馬鈴薯的主要病害之一。本研究采用PEG誘導(dǎo)原生質(zhì)體轉(zhuǎn)化的方法,借助ITS序列用GFP基因標(biāo)記馬鈴薯炭疽病菌后觀察其侵染過(guò)程,研究馬鈴薯在炭疽病菌脅迫下基因的差異表達(dá)情況,取得以下結(jié)論:1、馬鈴薯炭疽病菌的綠色熒光標(biāo)記利用定點(diǎn)突變改造馬鈴薯炭疽病菌ITS序列后,構(gòu)建了兩側(cè)含有ITS序列的GFP標(biāo)記載體pITGH,通過(guò)同源重組法將hyg B::gfp基因標(biāo)記到球炭疽菌的ITS序列上,獲得穩(wěn)定表達(dá)的轉(zhuǎn)化子C-106、C-163和C-168,提取轉(zhuǎn)化子的基因組DNA,經(jīng)PCR擴(kuò)增后,電泳檢測(cè)到400bp的gfp基因預(yù)期片段,經(jīng)測(cè)序表明gfp基因已整合至球炭疽菌基因組DNA中。2、轉(zhuǎn)化子的生物學(xué)特性采用常規(guī)方法測(cè)定轉(zhuǎn)化子的生物學(xué)特性,發(fā)現(xiàn)綠色熒光蛋白gfp基因的轉(zhuǎn)化對(duì)球炭疽菌的菌落形態(tài)、生長(zhǎng)速度和致病性等無(wú)影響,pH值對(duì)轉(zhuǎn)化子的菌絲生長(zhǎng)無(wú)影響,轉(zhuǎn)化子在pH值為4~12的PDA培養(yǎng)基上均可生長(zhǎng),最適pH值為8。3、球炭疽菌對(duì)馬鈴薯的侵染過(guò)程將熒光標(biāo)記菌株C-168接種于馬鈴薯莖稈和塊莖,分別取樣于熒光顯微鏡下觀察其侵染馬鈴薯的過(guò)程,發(fā)現(xiàn)球炭疽菌菌絲在寄主莖稈組織中沿著細(xì)胞壁向相鄰細(xì)胞進(jìn)行延伸,且在維管束細(xì)胞中延伸速度最快,其次為周皮細(xì)胞和表皮細(xì)胞,在髓部細(xì)胞中最慢;在馬鈴薯塊莖細(xì)胞中,菌絲沿著細(xì)胞壁向四周相鄰細(xì)胞延伸,隨著侵染時(shí)間的延長(zhǎng),菌絲交織呈網(wǎng)狀裂解細(xì)胞結(jié)構(gòu),不斷沿著細(xì)胞間隙向周圍細(xì)胞延伸。4、馬鈴薯在球炭疽菌脅迫下的基因表達(dá)譜分析將球炭疽菌接種于馬鈴薯后,采用RNA-Seq技術(shù)進(jìn)行測(cè)序,發(fā)現(xiàn)共有2936個(gè)基因發(fā)生差異表達(dá),其中上調(diào)基因1 492和下調(diào)基因1 444。差異表達(dá)基因經(jīng)過(guò)注釋后GO功能分為3類,在生物學(xué)過(guò)程(biological process)中主要有刺激反應(yīng)、應(yīng)激反應(yīng)和碳水化合物代謝過(guò)程等;在分子功能(Molecular Function)中主要涉及水解酶活性-作用于糖基鍵,裂解酶活性和四砒咯結(jié)合等;在細(xì)胞組成(cellular component)中主要有細(xì)胞外圍、外區(qū)和外部封裝結(jié)構(gòu)等。差異表達(dá)基因顯著性富集于24條Pathway,主要通路包括代謝途徑、苯丙烷代謝途徑、次生代謝產(chǎn)物的生物合成途徑、光合作用-天線蛋白及淀粉和蔗糖代謝途徑等。分析獲得19個(gè)連續(xù)性共表達(dá)差異基因參與顯著性富集代謝途徑,包括次生代謝產(chǎn)物的生物合成、苯丙烷代謝途徑和淀粉和蔗糖代謝途徑等,均表現(xiàn)為下調(diào)表達(dá)。植物與病原物互作途徑為非顯著性富集代謝途徑,涉及的差異表達(dá)基因有158個(gè),其中有5個(gè)為連續(xù)性共表達(dá)的差異基因,1個(gè)上調(diào)表達(dá),2個(gè)下調(diào)表達(dá),2個(gè)先下調(diào)再上調(diào)表達(dá)。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.32

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本文編號(hào)：2184068