色素万寿菊褐斑病防治及类甜蛋白基因克隆研究

发布时间：2018-10-26 18:27

【摘要】：色素万寿菊(Tagetes erecta)花朵可以提取天然色素“叶黄素”,广泛应用于医药、保健、食品等行业,因此具有较高经济价值；同时,色素万寿菊是一种观花期较长的花卉,大面积种植具有较好的景观效果,带动当地生态旅游产业的发展。基于上述两点,色素万寿菊已成为北京市延庆县四海镇支柱花卉产业之一。但是,由于连作年限的增加和雨季来临时沟域内空气湿度较大,导致四海色素万寿菊病害频发,严重影响产量和景观效果。本论文采取病害田间调查,病原物分离与鉴定,室内抑菌实验,田间药剂防治试验、生态套种试验、抗病基因克隆等研究方法,拟研制出减轻色素万寿菊褐斑病发生的适宜药剂防治配方、生态套种技术；同时克隆出类甜蛋白基因,为未来通过基因改良等手段提高色素万寿菊抗病性奠定基础。研究结果如下：病害田间调查发现：四海色素万寿菊前期主要病害为病毒病,根腐病,疫病,褐斑病；后期病害主要为褐斑病。生产过程中,褐斑病对产量和景观效果影响最为明显。从发生褐斑病的色素万素菊上采集病叶,分离并纯化病原物菌株。通过显微观察初步鉴定为链孢属(Alternari sp.);通过ITS分子生物学技术,准确鉴定为极细链格孢(Alternaria tenuissima)。系统树分析显示,极细链格孢与其他种链格孢有一定遗传关系、也有明显区别。室内抑菌实验发现：代森锰锌、世高、异菌脲、丙环唑、福星等药剂具有较好抑菌效果。田间药剂防治试验发现：“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%丙环唑2000倍液+70%代森锰锌750倍液”的防治效果较好；“枯草芽孢杆菌600倍液”也具有较好的防治效果,这为褐斑病生物防治及色素万寿菊生态安全生产提供了线索。生态套种试验发现：在四种不同的套种模式中,“万寿菊-荷兰土豆”生态套种的经济价值最高,病害减轻效果最明显,观赏时间延长、观赏效果增加,是最适宜推广的生态套种模式。运用PCR和RACE技术,从万寿菊中分离出类甜蛋白基因(TLP)。该基因全长cDNA 880bp,开放阅读框编码223个氨基酸。生物信息学分析表明：万寿菊类甜蛋白氨基酸序列的分子量为23933.8 Da；理论等电点6.51；分子式为C1051H1587N295O327S18;总原子量为3278;消光系数(280mm)为26440；脂肪系数：54.22；属稳定蛋白。在N-端具有22个氨基酸构成的疏水性信号肽和跨膜结构域。万寿菊类甜蛋白基因与向日葵PR5蛋白相似性为99%,与其他植物类甜白蛋白也有较高相似性。采用半定量技术,检测了TLP基因的表达变化。研究发现：健康植株中茎、叶、花组织中均检测不到TLP表达。植株被病害侵染后,茎、叶、花均能检测到TLP基因表达,花中TLP基因表达明显低其它器官。
[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.
【学位授予单位】：北京农学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S436.8;S681.9

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