【作者】 申珅；

【导师】 董金皋；

【作者基本信息】 河北农业大学 ， 植物学， 2013， 博士

【摘要】 大斑刚毛球腔菌（Setosphaeria turcica （Luttrell）Leonard﹠Suggs，俗称玉米大斑病菌）属子囊菌门毛座腔菌属，由其引起的玉米大斑病是一种严重威胁玉米生产的真菌病害，多发生于全球冷凉地域的玉米产区，常造成严重经济损失。研究表明，cAMP信号转导途径是真菌中普遍存在的细胞外信号跨膜转导途径，对植物病原真菌的形态发生及发育、次生代谢以及致病性等生物进程均起着非常重要的调控作用。本试验利用简并引物PCR法克隆了2个玉米大斑病菌cAMP磷酸二酯酶基因，其中包括1个高亲和力cAMP磷酸二酯酶基因（StH-PDE）和1个低亲和力cAMP磷酸二酯酶基因（StL-PDE）。分别创制StH-PDE和StL-PDE基因敲除突变体，对目的基因进行功能研究，主要研究结果如下：1.从玉米大斑病菌基因组中，获得了1个高亲和力cAMP磷酸二酯酶基因和1个低亲和力cAMP磷酸二酯酶基因，分别命名为StH-PDE和StL-PDE。其中，StH-PDE基因DNA全长3208bp，含有6个外显子和5个内含子，cDNA为2898bp，编码965个氨基酸，编码产物计算分子量约为107.16kD；StL-PDE基因DNA全长5054bp，含有5个外显子，4个内含子，cDNA为3089bp，编码1019个氨基酸。上述基因的内含子基本符合GT-AG法则。2. StH-PDE编码产物的氨基酸序列与多种病原真菌，如Aspergillus fumigatus、Botrytis cinerea、Metarhizium acridum、Neosartorya fischeri等的H-PDE氨基酸序列相似性在33.12%~36.60%之间，具有保守的Ⅰ型cAMP磷酸二酯酶催化结构域、Ⅰ型cAMP磷酸二酯酶保守位点和依赖金属离子的磷酸水解酶保守“HD”基序（HD motif），但与StL-PDE的相似性仅为20.33%，说明两个基因属于不同的磷酸二酯酶类群。StL-PDE编码产物的氨基酸序列与Trichoderma reesei、 Beauveria bassiana、Colletotrichum higginsianum、Talaromyced stipitatus等病原真菌的L-PDE氨基酸序列相似性为21.98%~23.06%，其中含有L-PDE特有的Ⅱ型cAMP磷酸二酯酶催化结构域。3.检测了StH-PDE基因在玉米大斑病菌不同发育时期的表达规律，发现StH-PDE基因在菌丝时期的表达量最高，在孢子萌发时期以及附着胞形成时期次之，在分生孢子发育以及侵染丝形成时期表达量最低。4.利用质粒pUCATPH和pBS，根据基因同源重组原理，构建了StH-PDE基因敲除载体。将重组DNA片段通过PEG介导转化玉米大斑病菌原生质体，经潮霉素筛选获得了潮霉素抗性转化子，通过潮霉素磷酸转移酶基因特异性引物及StH-PDE基因特异性引物对转化子进行PCR筛选，得到了9株阳性转化子；对其中3株转化子进行单孢分离，并进行Southern blot及RT-PCR验证，最终获得2株StH-PDE基因缺失突变体菌株△StH-PDE2和△StH-PDE3。对△StH-PDE2和△StH-PDE3突变体进行了功能分析，发现2株突变体菌落颜色呈深黄色，气生菌丝减少，且生长速度较野生型菌株慢；菌丝细胞膨大，细胞分隔变短且表面出现不规则褶皱，细胞壁完整性降低，菌丝细胞外包裹红色分泌物；其次，突变体菌株具产孢缺陷，菌丝可在玻璃纸表面诱导形成附着胞，但附着胞形成过程明显延迟，且穿透玻璃纸的能力降低；再者，突变体菌株细胞内甘油含量升高，抗盐胁迫能力增强，疏水性下降，易出现可湿表型，胞内黑色素含量降低；漆酶及HT-毒素活性没有明显变化；此外，经组织病理学观察发现，突变体菌株能够正常侵入寄主组织，但侵染效能较野生型有所降低。RT-PCR分析表明，StH-PDE基因对细胞壁合成相关基因（CHS、GFA、FKS、GSC、GEL）、微管蛋白基因Tubulin、疏水性调控基因MPG1、水甘油通道蛋白基因FPS以及调控黑色素合成的转录因子基因SMR的表达均有重要的调控作用。6.检测了StL-PDE在玉米大斑病菌不同发育时期的表达规律，结果表明，StL-PDE基因在玉米大斑病菌菌丝时期的表达量最高，其次是在孢子萌发时期和附着胞形成时期。7.利用相同的基因敲除载体构建策略构建了StL-PDE基因敲除载体，经潮霉素抗性和PCR筛选获得了10个转化子。对其中的△StL-PDE1和△StL-PDE10突变菌株单孢分离，并经Southern blot及RT-PCR验证，明确其为StL-PDE缺失突变体。对△StL-PDE1和△StL-PDE10突变体菌株进行了表型分析，发现突变体气生菌丝较野生型茂密，且生长速度较野生型菌株略快；突变体菌丝出现多分枝现象，分生孢子产量降低，且细胞分隔处表面出现不规则突起，菌丝可在玻璃纸表面诱导产生附着胞，但其穿透玻璃纸的能力较野生型降低；其次，突变菌株细胞内甘油含量积累升高，抗盐胁迫能力增强；胞内黑色素含量减少；漆酶活性显著降低。RT-PCR分析表明，△StL-PDE1、△StL-PDE10突变体中细胞壁合成相关基因（CHS、GFA、FKS、GSC、GEL）、微管蛋白基因Tubulin、疏水性调控基因MPG1和野生型菌株没有明显差异；水甘油通道蛋白FPS表达量略有下调。由此得出结论：1）StH-PDE基因在调控玉米大斑病菌菌丝细胞形态建成以及细胞完整性、分生孢子发育、次生代谢、感知营养的能力、胞内黑色素生物合成、应答盐胁迫反应以及致病性等方面均具有重要调控作用；2）StL-PDE在调控玉米大斑病菌分生孢子发育、菌丝附着胞的侵染能力以及胞内漆酶活性等方面具有重要调控作用。3）StH-PDE基因在玉米大斑病菌生长发育过程中担负主要的调控作用，而StL-PDE基因则担负较为次要的调控作用。更多还原

【Abstract】 Setosphaeria turcica which causing Northern Corn Leaf Blight, is one of importantphytopathogenic fungi, and always results in significant corn yield losses. cAMP signaltransduction pathway is a widespread extracellular signal transduction pathway in fungi,and playes important roles in regulating the growth, morphogenesis, development,secondary metabolism and pathogenicity of phytopathogenic fungi. In this research, twokey enzyme genes of Setosphaeria turcica, involved in cAMP signal transduction pathway,were cloned and named as StH-PDE and StL-PDE respectively. StH-PDE was encodedhigh-affinity cAMP phosphodiesterase and StL-PDE was encoded low-affinity cAMPphosphodiesterase. Functional analysis of StH-PDE and StL-PDE genes were explored bycreating the gene-knockout mutants. Main results in this paper were as follows:1. One high-affinity cAMP phosphodiesterase gene (StH-PDE) and one low-affinitycAMP phosphodiesterase gene (StL-PDE) were cloned with the candidate gene cloningstrategy. Among them, the full length DNA and cDNA of StH-PDE and StL-PDE had beenobtained. StH-PDE included3208bp DNA sequence with2898bp coding region andconsisted of6exons and5introns, and its predicted protein contained965aa with amolecular weight of107.16kDa. StL-PDE gene included5054bp and was interrupted byone intron and its ORF of3089bp was encoded1019amino acid residues and wasinterrupted by4intons. All introns were accordance with GT-AG rules.2. The low identity for the StH-PDE on nucleic acid level found for the high-affinitycAMP phosphodiesterase from Aspergillus fumigatus, Botryotinia fuckeliana, Metarhiziumacridum, Neosartorya fischeri was between33.12%and36.60%. PPDEase_Ⅰ3’5’ cyclicnucleotide phosphodiesterase, catalytic domain and metal dependent phosphodiesterase,HD/domain, and PDEase_Ⅰ3’5’ cyclic nucleotide phosphodiesterase, conserved site wasfound, whereas high-affinity cAMP phosphodiesterase was20.33%sequence similarity tolow-affinity cAMP phosphodiesterase of S. turcica. It was probable that two genesbelonged to two groups of phosphodiesterase and shared different catalyse substrate. Thelow identity for the StL-PDE on nucleic acid level found for the low-affinity cAMPphosphodiesterase from Trichoderma reesei, Beauveria bassiana, Colletotrichumhigginsianum, Talaromyced stipitatus was between21.98%and23.06%. PDEase_Ⅱ3’5’ cyclic nucleotide phosphodiesterase domain and translation elongation factor EFIB,gamma chain, conserved site was found in StL-PDE.3. StH-PDE mRNA expression level was determined in the different developmentstage of S. turcica. The result showed that StH-PDE mRNA expression level was thehighest in mycelium and lowest in conidium and infection hypha.4. The StH-PDE gene-disruption vector was constructed based on the genedouble-cross homologous combination theory and PEG-mediated gene transformationsystem. Nine transformants named as△StH-PDE1~△StH-PDE9were screened byhygromycin B and PCR with specific primers corresponding to hygromycinphosphotransferase gene and StH-PDE gene.△StH-PDE2,△StH-PDE3and△StH-PDE8were obtained by Southern blot analysis performed with the DIG-labeled HPHgene and StH-PDE gene as probes respectively. Furthermore, single spore of△StH-PDE2and△StH-PDE3were isolated and verification by RT-PCR. The function analysis wasindicated that mutants were dark yellow, showed different color with the wild type strain.The number of aerial hypha and the growth in mutants was reduced. The cell of hypha wasswollen and the compartmentation of the cell was shorter. The surface of the hypha wasdisplayed irregular lined. The cell wall integrity was destroyed. The red secretion wasadhered on the surface of the hypha. The sporulation defect was showed in mutants, whilehypha could germinated and formed appressorium associating gwith postphonement,furthermore, the penetrate ability on the cellophane surface was declined. Theaccumulation of intra-cellular glycerin content mutants was increased and showedreinforced salt-stress resistance. The hydrophobicity of mutants was impaired andpresented an wettable phenotype. The HT-toxin activity of mutants was resemble with wildtype strain. The pathogenicity test showed that mutants could penetrate on the host, butcouldn’t result in lession. The mRNA expression level of CHS、GFA、FKS、GSC、GEL、Tubulin、MPG1and FPS in mutants was lower than wild type strains in different extent.5. StL-PDE mRNA expression level was determined in the different developmentstage of S. turcica. The result showed that StH-PDE mRNA expression level was thehighest in mycelium and lower in spore germination and appressorium formation.6. The StL-PDE gene replacement vector was also constructed based on the samestrategy as StH-PDE. StL-PDE transformants named△StL-PDE1and△StL-PDE10wereisolated and verificated by PCR、southern blot and RT-PCR. The function analysis ofmutants exhibited increased aerial hypha formation and second-infect hypha branching,reduced conidiation, lower penetration ability on the cellophane surface, enhancedaccumulation of intra-cellular glycerin content, reinforced salt-stress resistance, reducedmelanin content and declined laccase activity. The PT-PCR analysis indicated that mRNAexpression level of CHS、GFA、FKS、GSC、GEL、Tubulin and MPG1was no differencein the wild type and mutant strains. Only the mRNA expression level of FPS displayed a little declined in the mutant strains.The above results can summarize the function of these genes:1) StH-PDE gene wasinvolved in cell wall integrity, sporulation, second metabolism, sense to nutrition, melaninbiosynthesis, salt-stress resistant and pathogenicity in S. turcica respectively.2) StL-PDEgene played an important role in the regulation of sporulation, penetration ability of thehypha and laccase activity in Setosphaeria turcica.3）StH-PDE gene might have apredominant role and StL-PDE gene played a secondary role in the regulation of growthand development n S. turcica.更多还原