【作者】 刘晓妹；

【导师】 郑服丛；

【作者基本信息】 海南大学 ， 分子植物病理， 2012， 博士

【摘要】 巴西橡胶树是重要的热带作物之一,其产品天然橡胶与钢铁、石油、煤炭并列为四大工业原料。多主棒孢菌[Corynespora cassiicola（B.&C.）Wei]是重要的植物病原真菌,可为害300多种植物,由其侵染引起的巴西豫胶树棒孢霉落叶病是影响天然橡胶生产的主要病害之一。目前国内外对该病害的研究集中在病原菌生物学、病害流行学和防治技术等方面,对病原菌致病机理,特别是致病相关功能基因及其调控等方面的研究很少。以巴西橡胶树棒孢霉落叶病作为研究对象,开展病原菌致病相关功能基因及其调控等方面的研究,对揭示该病原真菌的致病机理、了解该病原菌与寄主之间的互作机制具有重要的学术价值,同时也有助于我们发展新的防治措施。本研究工作运用基因同源克隆技术,从巴西橡胶树棒孢霉落叶病菌（C.cassiicola）中克隆了蛋白激酶（MAPK）同源基因CCKl和毒素编码基因ct的全长序列,通过潮霉素基因hygB插入突变和PEG介导的原生质体转化,获得了它们的基因敲除突变体,并对其表型做了测定。主要结果如下：1.根据真菌中已发现的MAPK蛋白的保守序列设计一对简并引物,从C.cassiicola基因组DNA中扩增获得了一段PMKl类MAPK同源基因片段,利用SEFA-PCR技术经两步扩增获得了该片段的5’端（1802bp）和3′端（652bp）侧翼序列,拼接校正后的DNA全长2710bp,命名为CCKl。将所得CCKl的DNA序列与GenBank中PMK1类MAPK同源基因的cDNA序列比对后,从内含子外侧设计引物,以C.cassiicola基因组RNA为模板经RT-PCR扩增获得了一段1065bp的cDNA片段,比对已获得的CCKl基因的DNA和cDNA序列,发现起始密码子ATG位于第1227～1229bp处,附近序列符合Kozak规则,即ACCATGT,终止密码子TAA位于第2502～2504bp处,自ATG到TAA的DNA全长1278bp,其中含5个外显子和4个内含子,外显子大小分别为110bp、196bp、382bp、348bp和29bp（位于第1227～1336,1390～1585,1640～2021,2072～2419和2476～2504碱基处）,内含子大小分别为53bp、54bp、50bp和56bp（位于第1337～1389,1586～1639,2022～2071和2420-2475碱基处）；推测开放阅读框编码354个氨基酸残基,分子量约40.98kDa,等电点（PI）为6.43,是一种微酸性蛋白质。经过在GenBank数据库搜索CCK1的相似蛋白和系统聚类分析,发现CCK1与丝状真菌禾谷类叶枯病病菌（Phaeosphaeria nodorum）的MAK2（XP₀01793869.1）,油菜黑胫病病菌（Letosphaeria maculans）的MAPK（AAM89501.1）,稻胡麻斑病病菌Cochliobolusmiyabeanus)的BMKl（AB180104.1）,玉米小斑病病菌（Cochliobolus heterostrophus）的CHK1（AF178977.1）,稻瘟病病菌（Magnaporthe grisea）的PMK1（U70134.1）,番茄枯萎病病菌（Fusarium oxysporum f.sp. lycopersici） FMKl（AF286533.1）等PMKl类基因编码的蛋白高度同源,相似性分别为99%、99%、98%、98%、93%和93%,而且含有一个参与双重磷酸化作用的MAPK蛋白激活域TEY（182AA·184AA）和Ser/Thr蛋白激酶保守结构域。因此可以推测本试验获得的CCKl基因为C.cassiicola的PMKl类MAPK基因,序列已提交GenBank（GU373808.1）。2.采用在pMD18-T Vector的T克隆位点、Sph I和Sal Ⅰ酶切位点,先后依次插入CCKl基因5′端、3′端和潮霉素基因hygB三个片段,成功构建了C.cassiicola的CCKl基因敲除载体pFHB,从该载体上扩增一段插有hygB基因的重组CCKl基因片段,转化C.cassiicola原生质体,对所得转化子经过PDA平板初筛、PCR验证和Southern杂交证实,确定得到的编号为FHB4的菌株是多主棒孢菌蛋白激酶基因CCKl的敲除突变体。表型测定结果表明：与野生型菌株相比,CCKl突变体完全失去了产孢能力,菌落颜色由棕灰色变为墨绿色,紧贴培养基生长,气生菌丝显著减少,菌丝生长速率明显下降,菌丝刚直、分支和隔膜数减少,菌丝细胞中色素沉积多,颜色加深呈深褐色,原生质浓缩明显；菌丝更耐高温；对酸碱环境的最适范围变窄；对多种盐的渗透胁迫反应减弱；对多菌灵和甲基硫菌灵更敏感；用菌饼接种嫩叶完全不能致病,但用粗毒素测定致病性时却表现出弱致病力；产漆酶时间相对延后,但漆酶相对产量提高,纤维素酶的相对产量也明显提高；在菌丝生长方面,外源Ca2+的促进作用和cAMP的抑制作用对突变体的影响更大。说明蛋白激酶CCKl在巴西橡胶树棒孢霉落叶病菌菌丝的生长、发育、分化、色素的沉着,分生孢子、毒素、漆酶和纤维素酶的产生,对环境的适应性和对寄主的致病性方面可能起着重要的调控作用,而且有些调控作用可能是与钙信号或cAMP途径相互协调共同完成的。3.根据NCBI网站EF667973.1序列设计一对引物,用同样的方法从C.cassiicola中克隆了1个毒素基因ct,DNA全长2800bp,cDNA全长180bp,起始密码子ATG位于第802～804bp处,终止密码子TAA位于第1095～1097bp处,白ATG到TAA的DNA序列全长296bp,存在3个外显子和2个内含子,外显子大小分别为105bp、60bp和15bp（位于第802～906,974～1033和1083～1097碱基处）,内含子大小分别为67bp和49bp（位于第907～973和1034～1082碱基处）,推测开放阅读框编码59个氨基酸序列,分子量约5.92kDa,等电点（PI）为5.70,是一种酸性蛋白质,与C. cassiicola pro-cassiicolin（ABV25895.1）的氨基酸序列相似性达85%。因此初步确定本试验获得的ct基因是预期的C.cassiicola的毒素基因,序列已提交GenBank （GU373809.1）。4.用pMD18-T Simple Vector做启始载体,将含全部编码序列的-段毒素基因片段克隆在该载体的T克隆位点上,经Sal I酶切后反向插入hygB基因,成功构建了C.cassiicola毒素基因敲除载体pCTH。以该载体为模板,扩增获得一段插有hygB基因的重组ct基因片段,转化巴西橡胶树棒孢霉落叶病菌原生质体,对所得转化子经过PDA平板初筛、PCR验证和Southern杂交证实,确定得到的是多主棒孢菌毒素基因ct基因的敲除突变体。表型测定表明：与其野生型CC004相比,毒素突变体在菌落和菌丝形态、生长,产孢量、孢子萌发,菌丝生长对温度、pH要求,产生漆酶和纤维素酶的能力方面基本未发生明显的改变。用菌饼接种嫩叶,致病性虽有减弱但仍很强,用粗毒素测定致病性时症状却明显减轻。说明毒素是巴西橡胶树棒孢霉落叶病菌的一个重要的毒力因子,但不是唯一的因子。更多还原

【Abstract】 Hevea rubber （Hevea brasiliensis） was an important tropical crop, and its product was one of the four major industrial raw materials along with steel, petroleum, and coal. Corynespora cassiicola （Bert.&Curt.） Wei, an important plant pathogenic fungus, infected more than300kinds of host plants. The Corynespora Leaf Fall （CLF） caused by C. cassiicola in Hevea rubber was a major disease influencing the production of natural rubber. Currently, foreign and local studies mainly focused on pathogen biology and disease epidemiology as well as control. However, little research had been directed to pathogeneisis mechanisms, especially pathogen-related genes and their regulations. Resaerch on the pathogeneisis-related genes in Hevea-C. corynespora system would be of great academic value in terms of revealing the pathogenesis and understanding the interaction mechanism between C.corynespora and its hosts. Furthermore, the findings would be beneficial to developing new control methods.In this research, full-length sequences of CCKl gene, a MAPK homologous gene, and a toxin gene ct, encoding cassiicolin toxin, were obtained using homology-based cloning method from the genome of C. cassiicola isolated from Hevea rubber. The disrupted genes were designed by insertion mutation with the hygromycin B phosphotransferase gene （hygB）, and gene knockout mutants were obtained through protoplast transformation mediated by poly ethylene glycol （PEG）, and the phenotypes of disruptants were also determined. The results were mainly as follows:1. According to the conserved amino acid sequence in some filamentous fungal MAPKs, a pair of degenerate primers was designed, and a MAPK homologous gene fragment of PMKl was amplified from the genomic DNA of C. cassiicola. With SEFA-PCR（Self-Formed Adaptor PCR）, the5end （1802bp） and3end （652bp） segments flanking the sequences were obtained by two-step amplification. The spliced and corrected DNA was2710bp in size and named CCKl. After comparing the sequences of the CCKl DNA and the cDNA of the homologous gene PMKl, a pair of primers were designed from an external intron. A1065bp cDNA fragment was amplified from the genomic RNA of C. cassiicola by RT-PCR. The initiation codon ATG was found located within1227bp and1229 bp and the neighboring sequence conforms to the Kozak rule, which was the ACCATGT. The stop codon TAA was within2502bp and2504bp. The sequence from ATG to TAA was1278bp, including5exons with110bp,196bp,382bp,348bp, and29bp, respectively （being within the1227-1336,1390-1585,1640-2021,2072-2419, and2476-2504bases）, and4introns with56bp,54bp,50bp, and56bp, respectively （being within the1337-1389,1586-1639,2022-2071, and2420-2475bases）. The amino acid sequence of the open reading frame was inferred to be354, with a molecular weight of40.98kDa and an isoelectric point of6.43, being a slightly acidic protein. After searching for proteins similar to CCKl in the GenBank database and conducting a phylogenetic tree analysis, CCKl was found to be highly homologous to MAK2from Phaeosphaeria nodorum （XP₀01793869.1）, MAPK from Letosphaeria maculans （AAM89501.1）, BMK1from Cochliobolus miyabeanus （AB180104.1）, CHK1from Cochliobolus heterostrophus （AF178977.1）, and PMK1from Magnaporthe grisea, and FMK1from Fusarium oxysporum f.sp. lycopersici and other proteins of the PMKl-type proteins, with similarities of99%,99%,98%,98%,93%, and93%, respectively. Furthermore, a TEY （182AA-184AA） of the MAPK protein activation domain-participating dual phosphorylation and Ser/Thr protein kinase-conserved domain were included. Therefore, the CCKl gene acquired from C. cassiicola in this experiment was an MAPK gene of PMK1type. The sequence had been submitted to GenBank （GU373808.1）.2. The pMD18-T vector T cloning site, Sph I, and Sal I enzyme cutting sites were successively inserted into the CCKl gene5end fragment,3end fragment, and hygB gene, thus, successfully constructing the CCKl gene knockout vector pFHB. A recombined CCKl gene fragment from the knockout vector containing the hygB gene was amplified, and used to transform the C. cassiicola protoplast.After PDA agar plate screening, PCR verification, and Southern blot hybridization,the mitogen activated protein kinase gene CCKl knockout mutant was confirmed. Compared with the wild-type strain, the CCKl mutant losed sporulation capacity and the color of the colony changes from brown and gray to blackish green. Aerial mycelial mass significantly reduced, and mycelial growth rate obviously decreased. The hypha was firm and straight with less branches and septa, whereas the puce pigmentation intensifies with evident protoplasm concentration. The mycelium was more capable of withstanding high temperature and the optimal range of environment pH narrowed, whereas osmotic stress response to a variety of salts weakened. The fungus was more sensitive to carbendazim and thiophanate-methyl, as well. Meanwhile, the leaves of the rubber tree inoculated with mycelia plugs did not develop disease but the mutant demonstrates weak virulence when crude toxin was tested. Laccase production was relatively postponed with a relative increase in production. Cellulase production was significantly increased. The growth promoting effects of exogenous Ca2+and the growth inhibiting effects of exogenous cAMP on mycelial growth of the CCK1mutant were significantly higher than that of the wide type. All of the above demonstrated that the mitogen activated protein kinase CCK1might be play a vital regulatory role in mycelial growth, development, cell differentiation, and pigmentation, as well as conidium formation, toxin, laccaes and cellulase production, adaptability to the environment, and pathogenicity of C. cassiicola in Hevea rubber, and might be involve in mycelial growth together with Ca2+or cAMP signaling pathway.3. According to the cas gene sequence （EF667973.1） in NCBI, a pair of degenerate primers was designed, and a toxin encoding ct gene was amplified from the genomic DNA of C. cassiicola by the same method as the CCK1gene. The spliced and corrected DNA was2800bp in size and contained the entire180bp open reading frame. The initiation codon ATG was found located within802bp and804bp and the stop codon TAA was within1095bp and1097bp. The sequence from ATG to TAA was296bp, including3exons with105bp,60bp, and15bp, respectively （being within the802-906,974-1033, and1083-1097bases）, and2introns with67bp and49bp, respectively （being within the907-973and1034-1082bases）. The amino acid sequence of the open reading frame encoding was inferred to be59, with a molecular weight of5.92kDa and an isoelectric point of5.70, being an acidic protein. After searching for proteins similar to CT in the GenBank database, CT was found to be hightly homologous with pro-cassiicolin from Corynespora cassiicola （ABV25895.1） with similarities of85%. Therefore, the ct gene acquired from C. cassiicola in this experiment might be a toxin encoding gene. The sequence had been submitted to GenBank （GU373809.1）.4. The pMD18-T simple vector T cloning site were successively inserted into the toxin encoding ct gene fragment, and then the hygB gene were inserted in antisense orientation into the ct sequence to the engineened Sal Ⅰ site, thus, successfully constructing the ct gene knockout vector pCTH. A recombined ct gene fragment from the knockout vector inserted with the hygB gene was amplified, and used to transform C. cassiicola protoplast by the PEG method. After PDA agar plate screening, PCR verification, and Southern blot hybridization,the toxin encoding ct knockout mutant was confirmed. There were no obvious difference in the colony color, mycelia morphology and growth, sporulation quantity, conidial germination, requirement of temperature and pH, and laccase and cellulase production between the mutant and the wild type. Meanwhile, the leaves of the rubber tree inoculated with mycelia plugs can cause typical symptoms but demonstrate weak virulence. However, when crude toxin was tested for pathogenicity, it displayed weaker virulence. All of the above demonstrated that the toxin was a impotant virulence factor but not a unique factor in the pathogenicity of C. cassiicola in Hevea rubber.更多还原