【作者】 谷守芹；

【导师】 董金皋；

【作者基本信息】 河北农业大学 ， 植物病理学， 2007， 博士

【摘要】 由凸脐蠕孢菌引起的玉米大斑病是玉米生产上的重要病害之一，常造成严重经济损失。研究表明，MAPK信号转导途径是真菌中普遍存在的细胞外信号转导途径，并对植物病原真菌的生长、发育和致病性有重要的调控作用。本文利用候选基因法克隆了玉米大斑病菌中的3个MAPK基因(STK1、STK2、STK3)，分别与调控玉米大斑病菌的渗透胁迫调节、致病性与分生孢子发育、细胞壁合成等3种类型的已知MAPK基因有非常高的同源性。在获得玉米大斑病菌STK1基因突变体的基础上，进行了玉米大斑病菌STK1基因的表达研究，确定了STK1基因在玉米大斑病菌分生孢子发育和渗透胁迫调节方面的重要功能，为研究其他MAPK基因提供了思路和方法。经对玉米大斑病菌STK1基因反义诱导表达突变体的构建，不仅为该基因作为靶基因进行病害防治奠定基础，而且STK1基因正义诱导表达突变体的构建还将为该基因在其它物种中的转化以及研究该基因与其它基因之间的关系提供了材料。对ATMT突变体和黑色素缺失突变体中STK1基因表达的研究，证明了玉米大斑病菌调控生长和发育有着更复杂的机制。本文主要结果如下：1．利用候选基因法克隆了3个玉米大斑病菌MAPK基因，即STK1，STK2和STK3。其中STK1和STK2基因获得了基因的cDNA全长和DNA全长，STK3基因为一个片段，获得了该基因的部分DNA序列。STK1基因cDNA全长和DNA全长的获得使前期研究获得的STK1基因片段得到完整化，并对GenBank中登录的该基因进行了更新(登录号为AY849317)。2．对STK1、STK2和STK3基因的结构进行了分析。STK1基因DNA全长1506bp，cDNA全长1071bp，编码356个氨基酸，有9个外显子和8个内含子，外显子1071bp，内含子共435bp；STK2基因DNA全长1277bp，cDNA全长1059bp，编码352个氨基酸，含有5个外显子，4个内含子，外显子共1059bp，内含子共218bp；STK3基因片段长度为591bp，可能存在3个内含子，共172bp，4个外显子总长419bp，该基因的编码序列从该片段的第3位开始，编码区域为417bp，预测编码蛋白中含有139个氨基酸。试验中发现的所有内含子均符合GT-AG法则。3．对玉米大斑病菌STK1、STK2和STK3三个MAPK基因的同源性和功能进行了分析和比较，其中STK1基因可能主要与渗透胁迫和应激胁迫调节有关：STK2基因主要与病菌的致病性和孢子发育有关；STK3基因主要与细胞壁合成有关。为了明确STK1基因的表达量的变化，本试验获得了长度为1287bp的玉米大斑病菌β-微管蛋白基因片段，该序列与玉米小斑病菌的β-微管蛋白基因同源性均为100％。以该基因为内参，利用半定量RT-PCR技术，研究了高渗条件对玉米大斑病菌STK1基因表达的影响，在高渗处理8h内，STK1基因的表达量比较稳定，但超过8h后，STK1基因的表达量迅速下降。4．在高渗胁迫条件下，玉米大斑病菌的菌落形态、颜色、生长速度发生严重变化，但STK1基因突变体则变化不明显。5．利用原核表达载体pET28a(+)及原核表达宿主菌BL21构建了玉米大斑病菌STK1基因的原核表达载体STK1-pET28a(+)，并成功进行了STK1基因的原核表达分析。为STK1蛋白的分离和在细胞中的定位研究奠定了基础。Southern杂交结果表明，玉米大斑病菌的STK1基因在基因组中以单拷贝形式存在。6．在优化玉米大斑病菌原生质体制备和再生条件的基础上，根据基因同源重组原理构建了含有潮霉素磷酸转移酶基因和氨苄青霉素基因两个抗性标记的STK1基因敲除载体，通过PEG介导的原生质体转化，获得了246个抗性转化子，通过使用设计的潮霉素磷酸转移霉基因特异性引物及STK1基因特异性引物对转化子的筛选，得到了一个STK1基因突变体，命名为STM-35。STK1基因敲除突变体STM-35的菌丝灰白色、气生菌丝少，菌落低矮，菌落中部菌丝呈浸润状，菌丝细胞中色素沉积少、菌丝较透明，没有发现分生孢子的形成，提取的HT-毒素对感病寄主叶片的毒力显著下降，致病性分析表明，该突变体的致病性大大降低。7．基于pSOI质粒构建了玉米大斑病菌STK1基因的反义和正义诱导表达载体，获得了30个反义转化子，其中有5个转化子为反义抑制突变体，它们与STK1基因敲除突变体的表现基本一致。在构建玉米大斑病菌ATMT突变体库的基础上，对与STK1基因突变体在菌落和菌丝形态方面非常相似、也不产生分生孢子的ATMT突变体STAM-26，进行了STK1基因的表达分析，发现该突变体中STK1基因能够正常表达，说明调控玉米大斑病菌菌丝和分生孢子发育的机制非常复杂，并不是由某一条途径单独调控。结论推测，STK1基因对玉米大斑病菌菌丝的生长发育没有影响，但可能与菌丝中色素等物质的积累有关，并调控病菌分生孢子的发育、HT-毒素的活性和致病性。更多还原

【Abstract】 Northern Com Leaf Blight, caused by Setosphaeria turcica, is one of the most important diseases in corn planting area. MAPK, the universal signal tranduction pathway in fungi, is one of the key protein kinase in regulating the growth, development and pathogenicity of fungal pathogens. Three MAPK genes, STKI (Setosphaeria turcica mitogen activated protein kinase 1), STK2 (Setosphaeria turcica mitogen activated protein kinase 2), STK3 (Setosphaeria turcica mitogen activated protein kinase 3), were cloned. The phylogenetic analyses revealed that STK1 possibly regulated the osmosis regulation and spore production, STK2 was similar to known MAPK genes in regulating pathogenicity of fungal pathogen, and STK3 was related to cell wall integrity MAP kinase genes. The mutated STK1 gene recombination vectors were constructed to study the gene function, and the results confirmed the above phylogenetic analyses of STKI gene. This work will facilitate the studies on similar functional analyses of the other two MAPK genes in S. turcica, the composition of MAPK signal pathway, and the relation between different MAPK pathways. The antisense inhibition of STK1 gene was done to make the basis of gene-targeted disease control strategy, and the positive gene expression induction vector was constructed for the transformation of the gene and studies the relationship between different genes. The ATMT mutants with similar characteristics as STK1 mutants were screened and the STK1 gene expression in these mutants were analyzed. The normal expression of STK1 gene in these mutants has shown the complex regulation mechanisms1.Three MAPK genes, STK1, STK2, STK3, were cloned with the candidate gene cloning strategy. The cDNA and DNA full-length sequences of STK1 and STK2, a cDNA and DNA fragment of STK3 were acquired. STK1 gene was updated in GenBank(GenBank accession AY849317).2.The structure of STK1, STK2 and STK3 were revealed. STK1 included a 1506bp DNA sequence with a 1071 bp coding region, nine exons and eight introns. The predicated protein of STK1 gene had 356 aa. STK2 gene includes 1277bp with a 1059bp cDNA sequence, five exons, and four introns. The predicated protein of STK2 gene had 352 aa. The STK3 gene fragment was 591bp, with three possible introns (172bp) and four possible exons (419bp). All introns were accordance with GT-AG rules.3.Phylogenetic analysis had shown the three genes possibly belonged to three signal pathways: STK1, related to osmosis regulation, stress reaction and spore development; STK2, related to pathogenicity; STK3, related to cell wall integrity.Aβ-microtublin gene in S. turcica were cloned, sequenced and used as the controls in studying the expression of STK1 gene in high osmosis. The STK1 gene expression was stable within the first 8 hours aider the high osmosis treatment, but rapidly descended after 8 hours treatment.4.Under the high osmosis stress, the wild type isolate exhibited high changes in colony morphology, color, growth speed, but changes in the STK1 mutants were not obvious.5.A prokaryote gene expression vector of STK1 was constructed, pET28a(＋) vector, E. coli BL21, and the STK1 gene expression were confirmed by SDS-PAGE and Western blotting.Southern hybridization results have shown the STK1 gene had a single copy in the genome of S. turcica.6.The STK1 gene-disruption vector was constructed based on the gene homologous combination theory and PEG gene transformation system. A STK1 gene-disruption isolate was successfully screened from 246 transformants and named as STM-35 in the thesis. The STM-35 mutant exhibited grey colony, few aerobic mycelia, transparent hypha, cell lysis in the center colony, significantly reduced HT-toxin activity and pathogenicity, without conidial production.7.The sense and antisense STK1 gene expression induction vectors were constructed based on the plasmid pSOI, and 5 antisense STK1 gene mutants were screened from 30 transformants. The characteristics ofSTK1 antisense inhibition mutants were similar to the gene-disruption mutant STM-35.STAM-26, a mutant acquired by ATMT method, and three melanin-loss mutants induced by UV radiation, exhibit similar traits as STM-35. STK1 gene expression was undisturbed in the two mutants had predicated there were complex network regulating the growth and development in S. turcica.The above results can summarize the function of STK1: insignificant effect on mycelium development and growth but related to the pigment accumulation in hypha; key factor regulating the conidial development; related to the HT-toxin activity and pathogenicity.更多还原