【作者】 万英；

【导师】 黄云； 蒋伶活；

【作者基本信息】 四川农业大学 ， 植物病理学， 2008， 硕士

【摘要】 极细链格孢菌（Alternaria tenuissima）是一种植物病原真菌,对农业生产危害巨大,然而其在生防、环保等方面也有应用,因此从分子水平研究该菌的功能基因的调控机制,对进一步开发利用该菌具有重要意义。在真核微生物的遗传学研究中LEU2和HIS3基因是重要的营养标记,通过遗传学手段,我们筛选获得了极细链格孢菌AtLEU2及AtHIS3基因,并且研究了AtLEU2及AtHIS3基因互补酿酒酵母的功能。以G418抗性基因作为选择标记,构建了用于敲除极细链格孢菌AtLEU2及AtHIS3基因的质粒,建立了极细链格孢菌的遗传转化系统,为进一步研究AtLEU2及AtHIS3基因的功能奠定了坚实的基础,主要研究结果如下:1、将极细链格孢菌cDNA表达文库转化到酵母LEU2缺失突变株,用SD-LEU培养基进行筛选,获得了3个正常生长的酵母转化子。对转化子质粒进行测序分析,发现这些基因编码的蛋白质序列均与酵母LEU2（ScLEU2）基因编码的363个氨基酸同源（命名为AtLEU2,其编码的蛋白质命名为Atleu2p）,该基因编码的蛋白与植物病原菌小麦颖枯病CAB72262;灰葡萄孢霉EDN21270;玉蜀黍赤XP₃86851,以及水稻稻瘟病病菌XP₃67617所编码的β-苹果酸脱氢酶同源,相似性分别达到94%、74%、1%和69%,与酿酒酵母、粗糙链孢霉EAA33847相似性为70%和63%。2、将极细链格孢菌cDNA表达文库转化到酵母HIS3缺失突变株,在SD-HIS培养基上筛选,获得了2个正常生长的转化子。所得基因的开放阅读框（ORF）含有717个碱基,编码238个氨基酸,与酵母HIS3（ScHIS3）基因同源（命名为AtHIS3,其编码的蛋白质命名为AtHis3p）,AtHis3p与植物病原真菌链格孢BAF69039,小麦颖枯病EAT83561,灰葡萄孢霉EDN23196,玉蜀黍赤霉XP₃86269,以及水稻稻瘟病病菌XP₃67617中编码的咪唑甘油磷酸脱水酶同源,相似性分别达到100%,90%,68%,65%和63%;与生防菌哈茨木霉P34041相似性达64%;与酿酒酵母,粗糙链孢霉XP₉61386编码的蛋白相似性达64%和66%。3、分别选择以AtLEU2及AtHIS3基因上下游同源序列500 bp左右作为同源臂,在上游同源序列3’端连接G418抗性基因（以保证缺失突变株的检测及鉴定）,构建了用于敲除极细链格孢菌AtLEU2及AtHIS3基因的质粒,为在极细链格孢菌中敲除AtLEU2及AtHIS3基因奠定了坚实的基础,为进一步研究链格孢菌AtLEU2及AtHIS3基因的功能及作用机理提供了重要的试验材料。4、基于极细链格孢菌在含有G418（100μg/mL）的PDA培养基上不能生长的药物敏感性实验结果,建立了用G418抗性基因作为选择标记的遗传转化系统,从而为研究极细链格孢菌功能基因的敲除、缺失突变株鉴定提供了可靠的筛选标记,并为后续研究打下必要的基础。更多还原

【Abstract】 Alternaria tenuissima is a plant pathogenic fungi,it has enormous harm in agriculture,but also used in biological control,environmental protection,and so on.It is important to study the regulatory mechanism of functional gene in Alternaria tenuissima. In Eukaryotic microorganisms,LEU2 and HIS3 genes is an important marker of nutrition. In this study,we screened the Alternaria tenuissima cDNA expression library in auxotroph yeast,and identified and characterized the A.tenuissima AtLEU2 and AtHIS3 genes.A recombinant vectors for knocking out AtLEU2 and AtHIS3 genes was constructed,and inserted the G418 resisitence gene as selection markers.This provide a basis for further study on functions of AtLEU2 and AtHIS3 genes.The main results obtained in this study are as fellows:1.Alternaria tenuissima cDNA expression library was tansformed into yeast strain with LEU2 deletion mutant,and there were 3 transformants could grow on SD-LEU medium.The result of DNA sequencing indicated that the 3 cDNA inserts contained the same open reading frame encoding the A.tenuissima homologue of ScLEU2（AtLEU2） with 363 amino acids.Atleu2p shows 94%,74%,71%and 69%identities in the amino acid sequence with CAB72262（P.nodorum）,EDN21270（B.fuckeliana）, XP₃86851（Gzeae） respectively,and also has homology with beta-isopropylmalate dehydrogenases of XP₃67617（M.grisea）2.Alternaria tenuissima cDNA expression library was tansformed into yeast strain with HIS3 deletion mutant,2 transformants were obtained from SD-HIS medium.The cDNA insert have a size of 717 bp in length,which encodes a protein of 238 amino acids. It was homologue of Sc tHIS3,the protein encided by AtHIS3 was named as AtHis3p. AtHis3p also shows 100%,90%,68%,65%,63%,64%and 66%identities with BAF69039（A.alternata）,EAT83561（P.nodorum）,EDN23196（B.fuckeliana）, XP₃86269（G.zeae）),XP₃67617（M.grisea）,P34041（T.harzianum）) and XP₉61386（N.crassa） respectively.3.We constructed recombinant vectors for knocking out AtLEU2 and AtHIS3 genes through DNA recombination,which contains about 500 bp upstream and downstream DNA fragments of AtLEU2 and AtHIS3 genes with the G418 resisitence gene inserted between them.This provides a basis for further study on functions of these genes in A. tenuissima.4.Drug sensitivity tests indicates that A.tenuissima could not grow on YPD plates containing G418（100μg/ml）.The genetic transformation system was established base on the test,which used G418 resisitence gene as selection marker.更多还原