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灰葡萄孢T-DNA插入突变菌株的致病性分析

标题: 灰葡萄孢T-DNA插入突变菌株的致病性分析
英文标题: Pathogenecity Analysis on One T-DNA Insertion Mutant Isolate of Botrytis Cinerea
作者: 张玉净
出版时间: 2011-01-01
所在大学: 河北农业大学
关键词: 灰葡萄孢,细胞壁降解酶,TAIL-PCR,RT-PCR,毒素
英文关键词: Botrytis cinerea,Cell wall degrading enzymes,TAIL-PCR,RT-PCR,Toxin
论文级别: 硕士
学位: 学位论文
导师: 张金林%董金皋
专业: 微生物与生化药学
提交时间: 2011
摘要: 灰霉病是一种由灰葡萄孢引起的世界性重要病害,灰葡萄孢寄主范围广,可侵染多种经济作物、观赏性植物,并造成严重经济损失.灰葡萄孢主要以菌丝、菌核、孢子形式在病残体或者土壤中越冬.由于灰葡萄孢繁殖快、遗传变异大、适合度高,使其防治一直难以解决.目前国内外仍主要采取化学农药,辅以生物及生态、农业等多种措施防治灰霉病.本研究从前期获得的400余株灰葡萄孢ATMT转化子中筛选到一株致病突变菌株BCG-183,经活化培养,从形态学水平对该菌株进行了表型分析,发现该菌株菌丝产量较野生型明显增大;在PDA平板上培养7 d后,菌落呈白色,明显区别于野生型菌株的灰色菌落,不产生分生孢子.将野生型菌株BC-22和突变菌株BCG-183分别接种到经表面消毒的番茄叶片和果实上,保湿培养3 d后发现突变菌株形成的病斑面积明显大于野生型菌株,说明突变菌株致病力较野生型明显增强.突变菌株在产细胞壁降解酶培养基中振荡培养6 d,利用DNS法测定酶活,发现产生的细胞壁降解酶酶活性强于野生型.将黄瓜种子和番茄种子在突变菌株和野生型菌株产毒培养滤液浸泡1 d后,于含有湿润滤纸的保湿培养皿中培养5 d发现突变菌株产毒培养滤液浸泡过的黄瓜和番茄种子萌发率显著下降;提取突变菌株和野生型菌株的粗毒素,将等量粗毒素接种于番茄叶片上,发现突变菌株粗毒素形成的病斑面积较野生型大,突变菌株的产酸能力较野生型明显增强.利用CTAB法提取突变菌株BCG-183的基因组DNA,利用TAIL-PCR技术扩增该菌株的T-DNA插入位点侧翼序列,从分子水平确定T-DNA插入位点.对序列进行生物信息学分析,发现T-DNA插入位点为灰葡萄孢基因组超级重叠群42(Supercontig 42)中假定蛋白编码基因(BC1G07455)的3'末端,破坏了该基因的表达.BC1G07455基因的cDNA全长1 221 bp,编码了406个氨基酸残基的假定蛋白,该蛋白与马尔尼菲青霉菌的水杨酸羟化酶、曲霉属的FAD依赖氧化还原酶、黄萎病菌的犬尿氨酸3-单加氧酶KMO(kynurenine 3-monooxygenase)同源性均为97%以上.RT-PCR分析表明,BC1G07455影响果胶酶基因Bcpg1、产毒基因BcBOT2的表达情况,使得Bcpg1、BcBOT2在突变菌株中表达上调,说明BC1G07455可能对果胶酶和毒素的产量起负调控作用.
英文摘要: The plant pathogenic fungus Botrytis cinerea causes grey mold of various crops and ornamental plants, which can cause serious economic lossing. B. cinerea belongs to Deuteromycotina and can live through the winter in sick deformed or soil by the mycelium, sclerotia or spores form. Because of lacking resistant germplasm resources caused by fast reproduction, genetic variation and high parasitic fitness of B. cinerea, gray mold is difficult to be prevented. Chemical method which is the main method combines with biological and ecological measures, agricultural measures and other measures to prevent gray mold.In this study, a pathogenic mutant strain of BCG-183 was screened from more than 400 ATMT transformants of B. cinerea from the previous work and was analyzed from the morphological phenotype: the hypha of mutant was more than that of the wild type. After culturing in PDA plates 7 days, the colony of mutant was white and didn't produce spores, while the wild-type colony was gray and produced a large amount of spores; the mutant and wild-type strains were respectively inoculated onto sterile tomato leaves and tomato fruits, the mutant formed a larger lesion area than the wild-type after cultured 3d in the moisture environment, which indicated that the virulence of the mutant was enhanced compared with that wild-type; the mutant and wild-type colony respectively cultured in producing cell wall degrading enzyme medium 3 days by shaking, then the activity of the cell wall degrading enzyme was detected by spectrophotometer, and the result was that cell wall-degrading enzyme activity of the mutant was better than that of the wild-type. Cucumber seeds and tomato seeds, which were soaked 1 day in the toxigenic filtrate of the mutant and wild-type strain respectively, were fostered on the wet filter paper 5 days. The result was that the germination ratio of the cucumber seeds and tomato seeds were both inhibited by the toxigenic filtrate of the mutant. After the toxin extracted from the mutant and wild-type strains was dripped onto sterile tomato leaves, the lesion area formed by the mutant was larger than that of the wild-type; Mutant's acid production was significantly enhanced compared with wild-type.The genomic DNA of wild type and BCG-183 were extracted by CTAB method. Subsequently, TAIL-PCR was used to amplify the flanking sequence of T-DNA insertion site from its genome to determine the disrupted gene. We confirm that T-DNA had inserted into the 3'-end of the hypothetical protein gene (BC1G07455) locating in the genome Super contig 42 (Supercontig 42) of B. cinerea by bioinformatics analysis. The gene BC1G07455 is 1 221 bp, encoding 406 amino acids, and the putative protein showed more than 97%homology with the Penicillium salicylate hydroxylase [GenBank accession No. 7023390],the FAD dependent oxidation reductase of the Aspergillus [GenBank accession No.4983596] and kynurenine 3-monooxygenase of Verticillium wilt fungus [GenBank accession No. 9532153].The transcription level of endopolygalacturonase encoding gene Bcpg1 and BcBOT2 of the mutant was higher than that of the wild-type strains. The expression of Bcpg1 and BcBOT2 was upregulated by the disruption of BC1G07455. It was indicated that BC1G07455 might play a negative regulatory role in the endopolygalacturonase and toxin production.