【摘要】：色素萬壽菊(Tagetes erecta)花朵可以提取天然色素“葉黃素”,廣泛應用于醫(yī)藥、保健、食品等行業(yè),因此具有較高經(jīng)濟價值；同時,色素萬壽菊是一種觀花期較長的花卉,大面積種植具有較好的景觀效果,帶動當?shù)厣鷳B(tài)旅游產(chǎn)業(yè)的發(fā)展�；谏鲜鰞牲c,色素萬壽菊已成為北京市延慶縣四海鎮(zhèn)支柱花卉產(chǎn)業(yè)之一。但是,由于連作年限的增加和雨季來臨時溝域內空氣濕度較大,導致四海色素萬壽菊病害頻發(fā),嚴重影響產(chǎn)量和景觀效果。本論文采取病害田間調查,病原物分離與鑒定,室內抑菌實驗,田間藥劑防治試驗、生態(tài)套種試驗、抗病基因克隆等研究方法,擬研制出減輕色素萬壽菊褐斑病發(fā)生的適宜藥劑防治配方、生態(tài)套種技術；同時克隆出類甜蛋白基因,為未來通過基因改良等手段提高色素萬壽菊抗病性奠定基礎。研究結果如下：病害田間調查發(fā)現(xiàn)：四海色素萬壽菊前期主要病害為病毒病,根腐病,疫病,褐斑��；后期病害主要為褐斑病。生產(chǎn)過程中,褐斑病對產(chǎn)量和景觀效果影響最為明顯。從發(fā)生褐斑病的色素萬素菊上采集病葉,分離并純化病原物菌株。通過顯微觀察初步鑒定為鏈孢屬(Alternari sp.);通過ITS分子生物學技術,準確鑒定為極細鏈格孢(Alternaria tenuissima)。系統(tǒng)樹分析顯示,極細鏈格孢與其他種鏈格孢有一定遺傳關系、也有明顯區(qū)別。室內抑菌實驗發(fā)現(xiàn)：代森錳鋅、世高、異菌脲、丙環(huán)唑、福星等藥劑具有較好抑菌效果。田間藥劑防治試驗發(fā)現(xiàn)：“10%世高750倍液+60%1嘧菌脂3000倍液”和“10%世高750倍液+60%百泰1500倍液”防治效果最好；“20%丙環(huán)唑2000倍液+70%代森錳鋅750倍液”的防治效果較好；“枯草芽孢桿菌600倍液”也具有較好的防治效果,這為褐斑病生物防治及色素萬壽菊生態(tài)安全生產(chǎn)提供了線索。生態(tài)套種試驗發(fā)現(xiàn)：在四種不同的套種模式中,“萬壽菊-荷蘭土豆”生態(tài)套種的經(jīng)濟價值最高,病害減輕效果最明顯,觀賞時間延長、觀賞效果增加,是最適宜推廣的生態(tài)套種模式。運用PCR和RACE技術,從萬壽菊中分離出類甜蛋白基因(TLP)。該基因全長cDNA 880bp,開放閱讀框編碼223個氨基酸。生物信息學分析表明：萬壽菊類甜蛋白氨基酸序列的分子量為23933.8 Da；理論等電點6.51；分子式為C1051H1587N295O327S18;總原子量為3278;消光系數(shù)(280mm)為26440；脂肪系數(shù)：54.22；屬穩(wěn)定蛋白。在N-端具有22個氨基酸構成的疏水性信號肽和跨膜結構域。萬壽菊類甜蛋白基因與向日葵PR5蛋白相似性為99%,與其他植物類甜白蛋白也有較高相似性。采用半定量技術,檢測了TLP基因的表達變化。研究發(fā)現(xiàn)：健康植株中莖、葉、花組織中均檢測不到TLP表達。植株被病害侵染后,莖、葉、花均能檢測到TLP基因表達,花中TLP基因表達明顯低其它器官。
[Abstract]:Pigment (Tagetes erecta) flower can extract natural pigment "lutein", widely used in medicine, health care, food and other industries, so it has high economic value; At the same time, pigmented marigold is a kind of flower with long flowering period. Planting in a large area has better landscape effect and promotes the development of local ecotourism industry. Based on the above two points, pigment marigold has become one of the pillar flower industries in Sihai Town, Yanqing County, Beijing. However, due to the increase of continuous cropping years and the high air humidity in the gully area during the rainy season, there are frequent diseases of pigment marigold in the four seas, which seriously affect the yield and landscape effect. In this paper, disease field investigation, pathogen isolation and identification, laboratory bacteriostatic experiment, field insecticide control experiment, ecological interplanting experiment, disease resistance gene cloning and so on were studied. To develop a suitable chemical control formula and ecological interplanting technology to reduce the occurrence of pigment Marigold brown spot. At the same time, the sweet protein-like gene was cloned, which laid a foundation for improving the disease resistance of pigment marigold by gene improvement in the future. The results were as follows: the main diseases in the field were virus, root rot, blight, brown spot in the early stage and brown spot in the later stage. In the process of production, brown spot had the most obvious effect on yield and landscape effect. The diseased leaves were collected from pigment Vanilla, which caused brown spot, and the pathogenic strains were isolated and purified. Preliminary identification of Alternaria (Alternari sp.); by microscopic observation and identification of (Alternaria tenuissima). As Alternaria by ITS molecular biology technique Phylogenetic analysis showed that very fine Alternaria had a certain genetic relationship with other species of Alternaria. Laboratory bacteriostatic experiments showed that mancozeb, Shigao, isobarbazone, propiconazole and Foxin had better bacteriostatic effect. The results of field pesticide control experiments showed that the best control effect was "10% Shigao 750 times solution 601 pyridamole 3000 fold solution" and "10% Shigao 750 times solution 60100 Tai 1500 fold solution". The control effect of "20% pyrazole 2000 times solution 70% mancozeb 750 times solution" and "Bacillus subtilis 600-fold solution" also had better control effect, which provided clues for biological control of brown spot and ecological safety production of pigment marigold. The ecological interplanting experiment showed that among the four different interplanting models, the ecological interplanting of "marigold and Dutch potato" had the highest economic value, the most obvious effect of disease mitigation, the prolongation of ornamental time and the increase of ornamental effect. It is the most suitable ecological interplanting model. PCR and RACE techniques were used to isolate the sweet protein-like gene (TLP). From marigold. The full length of the gene is cDNA 880 BP, and the open reading frame encodes 223 amino acids. Bioinformatics analysis shows that the molecular weight of amino acid sequence of stevioprotein is 23933.8 Da;, the molecular formula is C1051H1587N295O327S18, the total atomic weight is 3278, the extinction coefficient (280mm) is 26440, the fat coefficient is 54.22, the molecular formula is C1051H1587N295O327S18, the total atomic weight is 3278, the extinction coefficient (280mm) is 26440, the fat coefficient is 54.22; It belongs to stable protein. A hydrophobic signal peptide and a transmembrane domain with 22 amino acids at the N-terminal. The similarity between Marigold stevioprotein gene and sunflower PR5 protein is 99g, and it is also similar to other plant sweet albumin. The expression of TLP gene was detected by semi-quantitative technique. It was found that TLP expression was not detected in stem, leaf and flower tissues of healthy plants. TLP gene expression was detected in stem, leaf and flower, and the expression of TLP gene in flower was significantly lower than that in other organs.
【學位授予單位】：北京農(nóng)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S436.8;S681.9

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