【作者】 冯飞；

【导师】 邱德文；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2007， 博士

【摘要】 链格孢菌既是一类重要植物病原真菌，又是具有应用前景的生物资源。从分子水平研究该类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。HOG途径参与诱导胁迫反应基因的表达、细胞形态恢复、信息素反应途径的阻遏及致病性等过程。Hog1p和Pbs2p蛋白激酶在细胞壁结构完整性、孢子分化、菌丝形成和浸入生长以及高渗透压甘油形成过程中起到重要作用。本研究通过运用基于λ噬菌体特异位点重组反应Gateway^?系统构建极细链格孢菌（Alternaria tenuissima）cDNA表达文库。通过遗传学手段，我们筛选获得了极细链格孢菌（A．tenuissima）AtHOG1及AtPBS2基因，并且研究了AtHOG1及AtPBS2基因的功能。建立了以抗性基因作为选择标记的技术体系，利用DNA同源重组技术，成功构建了用于敲除极细链格孢菌（A．tenuissima）AtHOG1及AtPBS2基因的质粒，为进一步研究AtHOG1及AtPBS2基因的功能奠定了坚实的基础，主要研究结果如下：1、链格孢属（Alternaria Nees）真菌是一类庞大的，难以进行形态鉴定的常见真菌。为了进一步确定本试验所用菌种的分类地位，本文成功克隆了rDNA基因。通过序列比对，构建了7个相似菌株的系统发育树，发现该菌株与极细链格孢菌（A．tenuissima）相应的rDNA高度同源，因此将链格孢菌JH505菌株定名为极细链格孢菌（A．tenuissima）。2、构建了含有attL1及attL2重组位点的重组表达质粒（pRS-DEST42），使用Gateway^? LR重组技术与入门文库发生特异重组交换而构建表达文库。经检测，表达文库的平均滴度为2.44x10⁶（cfu／ml），文库总容量为2.44x10⁷。阳性克性率为100％，平均插入片段大约为1381 bp左右。通过对阳性克隆测序结果的分析，cDNA序列完整。3、从7.62x10⁵个极细链格孢菌（A．tenuissima）cDNA表达文库酵母转化子中筛选获得了12个能使酵母hog1基因缺失突变株在YEPD+1MNaCl中生长且含有极细链格孢菌（A．tenuissima）cDNA表达文库质粒的转化子。这些基因编码的蛋白质序列均与酵母HOG1（ScHOG1）基因编码的355个氨基酸同源，命名为AtHOG1，其编码的蛋白质命名为AtHog1p，该基因编码的蛋白与稻瘟菌MG01822.4蛋白、黑粉菌UM02357.1蛋白、禾谷镰刀菌FG09612.1蛋白及酿酒酵母ScHog1p蛋白的相似性分别为96％、92％、90％和88％。序列比对结果显示，酵母ScHog1p蛋白的催化结构域与其同源蛋白具高度保守，而且三个催化位点及赖氨酸残基均位于ATP结合域内，说明Hog1p蛋白在丝状真菌中是高度保守的。4、从1.56×10⁵个转化子中最终分离而获得了1个能使酵母pbs2缺失突变株在高盐环境下生长的表达文库质粒。该基因全长2，492 bp，编码683个氨基酸。与酵母ScPbs2p蛋白具有42.6％的同源性，命名为AtPBS2，其编码的氨基酸为AtPbs2p。AtPbs2p与烟曲霉XP₇52961、稻瘟菌XP₃66048及禾谷镰刀菌XP₃88867同源序列分别具有55.3％、50.2％和48.6％的相似性。ScPbs2p的催化结构域在丝状真菌也是高度保守的。5、通过对极细链格孢菌（A．tenuissima）cDNA表达文库的筛选，获得能使酿酒酵母HOG1及PBS2基因缺失突变株具有对抗盐胁迫反应的质粒，通过对AtHOG1及AtPBS2基因在酵母突变菌株中的再次功能验证，发现AtHOG1及AtPBS2基因分别发挥了HOG通路途径的HOG1及PBS2基因的功能，即在盐胁迫条件下体现出与ScHog1p及ScPbs2p蛋白相同的功能，使酵母缺失突变株在高盐环境中生长。6、基于极细链格孢菌（A．tenuissima）在含有G418（100μg／ml）和潮霉素（Hygromycin B）（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抗性基因及潮霉素（Hygromycin B）抗性基因作为选择标记的技术体系，从而为研究极细链格孢菌（A．tenuissima）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。7、利用同源重组技术，分别选择以AtHOG1及AtPBS2基因上下游同源序列约500 bp作为同源臂，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。成功构建了用于敲除极细链格孢菌（A．tenuissima）AtHOG1及AtPBS2基因的质粒，为在极细链格孢菌（A．tenuissima）中敲除AtHOG1及AtPBS2基因奠定了坚实的基础，为进一步研究链格孢菌（A．tenuissima）AtHOG1及AtPBS2基因的功能及作用机理提供了重要的试验材料和工具。更多还原

【Abstract】 The high osmolarity and glycerol （HOG） pathway in eukaryotes involves in many processes, suchas expression and regulation of genes induced by drought stress、cell morphology、repression of sexpheromone response and pathogenicity. In this study, we constructed a Alternaria tenuissima cDNAexpression library based on the integrase-excisionase system of bacteriophageλ, screened the library,and identified and characterized the A.tenuissima AtHOG1 and AtPBS2 genes. In addition, weestablished that resistant genes for G418 and hygromycin B could be used as selection markers fordisruption of genes in A. tenuissima. Furthermore, we constructed plasmids for knocking out AtHOG1and AtPBS2 genes using DNA recombination technique, which would provide a basis for further studyon functions of AtHOG1 and AtPBS2 genes. The main results obtained in this study are as fellows:1, The genus Alternaria contains ubiquitous, saprophyte molds, which are difficult for speciesidentification base on morphology. The morphology of the Alternaria sp. strain JH505 isolated in ourlab is similar to that of A. radicina、A. brassicae、A. solani and A. brassicicola. In order to furtherconfirm taxonomic status of Alternaria strain JH505, the rDNA ITS sequence was PCR-amplified fromthe strain. GenBank Database Blast Search and phylogeny tree analysis results indicated that it is astrain ofA. tenuissima.2, A destination vector pRS-DEST42 containing attL1 and attL2 recombination sites wasconstructed. The entry cDNA library of A. tenuissima was transferred into the yeast destination vectorpRS-DEST42 through the LR recombination reaction with the Gateway^? LR Clonase enzyme mixture.As a result, the cDNA expression library has a titer of 2.44×10⁶ （cfu/ml） and a total clones of 2.44×10⁷.The rate of positive recombinants clones was 100% and the size of average insert cDNA was 1.38 kb.3, Twelve transformants of the yeast hog1 mutant could grow on YPD plates containing 1M NaCland contained cDNA clones. DNA sequencing indicated that the 12 cDNA inserts contained the sameopen reading frame encoding the A. tenuissima homologue of ScHOG1 （AtHOG1） with 355 amino acidsin length. AtHoglp shows 96%, 92%, 90% and 88% identities in the amino acid sequence with thehypothetical proteins MG01822.4 （Magnaporthe grisea）, UM02357.1 （Ustilago maydis）, FG09612.1（Fusarium graminearum）, and ScHoglp of S. cerevisiae, respectively. The catalytic domain in ScHoglpis highly conserved in its homologous proteins, and the three catalytic sites as well as the lysine residueinvolved in the ATP-binding in ScHoglp are also present in its homologues. These observations suggestthat Hoglp is highly conserved in these filamentous fungi.4, From 1.6×10⁵ library cDNA transformants we isolated one cDNA clone that could complementthe salt sensitivity of the yeast pbs2 mutant. The cDNA insert was sequenced to have a size of 2,492 bpin length, which encodes a protein of 683 amino acids with 42.6% sequence identity to that of ScPbs2p,which indicates this cDNA sequence encodes the A. tenuissima AtPbs2p. AtPbs2p also shows 55.3%,50.2% and 48.6% sequence identities with its sequence homologues of Aspergillus fumigatus （XP₇52961）, Magnaporth grisea （XP₃66048） and F. graminearum （XP₃88867）, respectively. Thekinase catalytic domain of ScPbs2p was conserved in its homologues of those filamentous fungi.5, Our study indicates that A. tenuissima could grow on YPD plates containing 1M NaCl. PlasmidcDNA from positive transformants were then individually reintroduced back to the hog1 and pbs2mutant to confirm their sodium-tolerance phenotypes, respectively. As a result, we confirmed that alltransformants contained cDNA clones conferring the sodium-tolerance phenotypes in the yeast hog1and pbs2 mutant. AtHOG1 and AtPBS2 genes complement the functions of ScHOG1 and ScPbs2p insodium tolerance, respectively.6, Drug sensitivity tests indicates that A. tenuissima could not grow on YPD plates containingG418（100μg/ml） and Hygromycin B（100μg/ml） in this study, so we established that resistant genes forG418 and Hygromycin B could be used as selection markers for transformation ofA. tenuissima.7, We constructed recombinant vectors for knocking out AtHOG1 and AtPBS2 genes through DNArecombination, which contains about 500 bp upstream and downstream DNA fragments of AtHOG1 andAtPBS2 genes with the G418 resisitence gene inserted between them. This provides a basis for furtherstudy on functions of these genes in A. tenuissima.更多还原