【作者】 曹志艳；

【导师】 董金皋；

【作者基本信息】 河北农业大学 ， 植物病理学， 2009， 博士

【摘要】 DHN黑色素在子囊菌和半知菌中广泛存在,具有保护微生物免受紫外线和溶菌酶的作用,也是某些植物和动物病原真菌的致病相关因子。研究表明,沉积在病菌附着胞壁上的黑色素,保证了病菌能够产生足够的膨压穿透寄主组织致病。本文利用候选基因法克隆了4个玉米大斑病菌(Setosphaeria turcica)黑色素合成途径相关基因,其中包括1个还原酶基因(St3HNR),2个漆酶基因(StLAC1和StLAC2)和1个调节基因(StMR),它们分别与已知丝状真菌的相应基因具有较高的相似性。试验利用同源重组的方法获得玉米大斑病菌St3HNR和StLAC1基因敲除突变体,通过RNAi技术获得StMR基因沉默转化子,在此基础上,对目的基因进行功能研究,主要研究结果如下:1.利用候选基因法克隆了玉米大斑病菌的1个1,3,8-三羟基萘还原酶基因,2个漆酶基因和1个调节基因,分别命名为St3HNR、StLAC1、StLAC2和StMR。获得了St3HNR、StLAC1和StMR基因的全长cDNA和DNA序列,StLAC2基因的部分DNA序列。将St3HNR基因全序列及蛋白序列提交GenBank,获得基因登录号为No. EU681832,蛋白登录号为No. ACD47140。2.对St3HNR、StLAC1和StMR基因结构进行了分析。St3HNR基因DNA全长918 bp,cDNA为804 bp,编码267个氨基酸,分子量约为28.28 kD,有3个外显子和2个内含子;StLAC1基因DNA全长1855 bp,cDNA为1806 bp,编码601个氨基酸,含有2个外显子,1个内含子;StMR基因DNA全长3276 bp,cDNA为3036 bp,编码1011个氨基酸,含有4个外显子,3个内含子。试验中获得的所有基因的内含子均符合GT-AG法则。3.对玉米大斑病菌St3HNR基因的相似性进行分析,结果表明,St3HNR基因编码产物与多种植物病原真菌的1,3,8-三羟基萘还原酶氨基酸序列具有95%以上的相似性,具有保守的还原酶结构域,但与玉米大斑病菌的1,3,6,8-四羟基萘还原酶(St4hnr)的相似性仅为45.9%,说明两个基因属于不同的还原酶类群,在黑色素合成途径中催化不同反应。StLAC1基因编码的氨基酸序列与多数真菌漆酶具有60%以上的相似性,StLAC2与Hortaea acidophila、Fusarium proliferatum、Gaeumannomyces graminis等真菌漆酶基因具有33%~46%的相似性,但StLAC1和StLAC2间相似性仅为26.34%;StLAC1编码的蛋白具有漆酶特有的3个Cu2+活性位点,即Cu-oxidise superfamily,并且活性区域高度保守。StMR基因编码的蛋白与水稻胡麻斑病菌(Bipolaris oryzae)、玉米小斑病菌(Cochliobolus heterostrophus)中调控黑色素合成的转录因子相似性为88%,该转录因子在N末端具有2个ZnF_C2H2结构和一个类似Zn(II)2Cys6的GAL4 DNA结合域,与水稻稻瘟病菌Pig1p、瓜类炭疽病菌Cmr1p等转录因子结构相同,且相应区域高度保守。4. Southern杂交结果表明,玉米大斑病菌的St3HNR和StLAC1基因在基因组中均以单拷贝形式存在,StLAC2基因以多拷贝形式存在。5.利用真核表达载体pPIC9K构建了玉米大斑病菌St3HNR基因表达载体pPIC9K-St3HNR,采用电击转化法将重组质粒转入表达宿主菌GS115中并进行了诱导表达,SDS-PAGE检测结果表明St3hnr蛋白在宿主菌中有分泌表达。6.利用质粒pUCATPH和pBS,根据基因同源重组原理,构建了St3HNR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St3HNR基因特异性引物对转化子进行PCR筛选,得到了6个St3HNR基因缺失转化子;以潮霉素基因和玉米大斑病菌St3HNR基因为探针分别进行Southern blot验证,最终确定6个转化子均为突变体,命名为△3hnr1、△3hnr2、△3hnr3、△3hnr4、△3hnr5和△3hnr6。对突变菌株进行表型分析,结果显示6株突变体菌落均成棕红色,菌丝、分生孢子透明,菌丝和菌落形态无明显变化,但生长速度略小于野生型菌株;突变菌株产孢能力、HT-毒素产生能力、分生孢子萌发和附着胞形成能力均无明显变化;但是突变菌株附着胞膨压、附着胞穿透能力、健康寄主上的致病力显著下降或丧失,突变菌株在创伤寄主上的侵染能力没有显著变化,但是病斑扩展面积较小。7.以ABTS为底物,采用分光光度计在420 nm下测定漆酶活力,确定玉米大斑病菌细胞内漆酶活力的影响因素。结果表明,酶活测定最佳反应条件为0.6 mmol/L ABTS底物和pH2.6的缓冲液,在反应开始后5 min进行测定;试验还发现,在250μmol/L Cu2+浓度下培养的玉米大斑病菌漆酶活性最强。8.利用相同的基因敲除载体构建策略构建了StLAC1基因敲除载体,潮霉素和PCR筛选后获得转化子StLAC1-M2、StLAC1-M3、StLAC1-M4。转化子与野生型菌落颜色没有明显差别,但是检测到StLAC1-M3和StLAC1-M4的胞外漆酶活性降低。9.利用PCR技术分两步扩增StMR基因片段,构建了RNAi基因沉默载体,通过潮霉素筛选得到了不同沉默表型的转化子。获得的6个转化子与野生型菌株相比,菌落颜色均发生了明显变化,由灰黑色变成了黄白色,但是不同转化子的颜色有差异。转化子R5、R7、R9和R8气生菌丝低矮,菌丝密度高,菌落边缘整齐。所有突变菌株均不产生分生孢子,菌丝透明,无黑色素沉积,菌丝形态不规整,多数弯曲并纠结在一起。由此得出结论:1)St3HNR基因在玉米大斑病菌黑色素合成、附着胞穿透力和致病力等方面具有重要功能;2)玉米大斑病菌中存在漆酶和至少2个漆酶的编码基因,但StLAC1基因与玉米大斑病菌黑色素合成关系不明显;3)StMR基因可以影响黑色素的合成,但具体调节方式还需进一步验证。以上基因功能的研究方法为黑色素合成途径中其它功能基因的研究提供了思路。更多还原

【Abstract】 DHN melanin is common in ascomycetous and imperfect fungi, which can protect microorganisms from UV light and the action of microbial lytic enzymes. It is considered as one of the important virulence factors for certain plant and animal pathogenic fungi. The roles of melanin have been studied extensively in regard to the melanization of the appressorial cell wall and the penetration process in phytopathogenic fungi. In this research, four genes of Setosphaeria turcica, involved in DHN melanin biosythnthesis pathway, were cloned and named as St3HNR, StLAC1, StLAC2 and StMR respectively. St3HNR encoded naphthalene reductase, StLAC1 and StLAC2 genes belonged to laccase gene, StMR encoded a transcription factor. All of them shared high amino acid homology with the corresponding proteins in other pathogenic fungi. Functional analysis of St3HNR, StLAC1 and StMR genes were explored by creating the gene-knockout mutants and RNAi mutants. Main results in this paper were as follows:1. One 1,3,8-Trihydroxynaphthalene(1,3,8-THN) reductase gene (St3HNR), two laccase genes (StLAC1 and StLAC2) and one regulation gene (StMR) were cloned with the candidate gene cloning strategy. Among them, we had cloned the full length DNA and cDNA of St3HNR, StLAC1 and StMR and also obtained partial coding region of StLAC2. The ORF and amino acid sequence of St3HNR had been deposited in the GenBank database and the accession numbers were No.EU681832 and No.ACD47140.2. Gene structures of St3HNR, StLAC1 and StLAC2 were analysised. St3HNR included 918 bp DNA sequence with 804 bp coding region and consisted of 3 exons and 2 introns. Its predicted protein contained 267 aa with a molecular weight of 28.28 kDa. StLAC1 gene included 1855 bp and interrupted by one intron and its ORF of 1806 bp encoded 601 amino acid residues. StMR gene with 3276 bp encoded a protein of 1011 amino acid residues and was interrupted by 3 introns. All introns were accordance with GT-AG rules.3. Homology analysis showed that the deduced amino acid sequence from the St3HNR gene shared 95% similarity to fungal 1,3,8-THN reductase and a complete reductase domain was found, whereas 1,3,8-THN reductase had 45.9% sequence similarity to 1,3,6,8-THN(St4hnr) of S. turcica. It was probable that two genes belonged to two groups of reductase and catalyse different substrate. StLAC1 exhibited the high identities to known laccases in fungi with over 60%. The low identity for the StLAC2 on nucleic acid level was found for the laccase from Hortaea acidophila, Fusarium proliferatum, Gaeumannomyces graminis with 33% ~ 46%. StLAC1 and StLAC2 genes exhibited much less homology identity (26.34%). Stlac1 protein has three conserved copper binding- regions, especially conserved regions with high identity. The deduced amino acid sequence of the StMR shared 88% similarity with the transcription factor of Bipolaris oryzae and Cochliobolus heterostrophus. The predicted S. turcica Stmr protein has two ZnF_C2H2 domains and one GAL4 domain as Zn(II)2Cys6 binuclear cluster close to its N terminus, similar to Magnaporthe grisea Pig1p and Colletotrichum lagenarium Cmr1p. Interestingly, all three domains proteins are highly similar.4. Southern hybridization results showed that genes of St3HNR and StLAC1 had single copy in the genome of S. turcica, whereas there are multicopy of StLAC2.5. Eukaryotic gene expression vector of St3HNR, pPIC9K-St3HNR, was constructed and expressed in host yeast strain GS115 through electroporation. The St3hnr protein expression was confirmed by SDS-PAGE.6. The St3HNR gene-disruption vector was constructed based on the gene double-cross homologous combination theory and PEG-mediated gene transformation system. Transformants were screened by hygromycin B and PCR with specific primers corresponding to hygromycin phosphotransferase gene and St3HNR gene. Six St3HNR gene-disruption mutants, named as△3hnr1、△3hnr2、△3hnr3、△3hnr4、△3hnr5 and△3hnr6, were obtained by Southern blot analysis performed with the DIG-labeled HPH gene and St3HNR gene as probes respectively. Although wild type strain and mutants were similar in form of colony and hypha, sporulation, HT-toxin activity, rates of spore germination and appressorium formation, all mutants exhibited brownish-red colony, colourless hypha and conidium, weakend turgor pressure and penetration of appressorium, slow vegetative growth rate and low pathogenicity on health host. The pathogenicity of mutants was restored while they were inoculated on wound corn leaves, but the area of lesion was decreased compared with wild type.7. The laccase activity of S. turcica was tested by spectrophotometer under 420 nm with ABTS as the substrate, and the influence of the test conditions on the enzyme activity were studied. The results showed that optimal reaction pH value, concentration of substrate and reaction time were 2.6, 0.6 mmol/L and 5 min respectively. In addition, the best laccase activity of S. turcica was screened when it grew on the medium containing 250μmol/L CuSO4.8. The StLAC1 gene replacement vector was also constructed based on the same strategy as St3HNR. StLAC1 transformants named StLAC1-M2, StLAC1-M3 and StLAC1-M4, which exhibited same grey colony as wide type. Extracellular oxidase activity of laccase in transformants StLAC1-M3 and StLAC1-M4 were reduced.9. The silencing vector encoding hairpin RNA of the StMR fragment was constructed in a two-step PCR based cloning, and introduced into the S. turcica genomic DNA. Six transformants with different color colony from less-pigmented transformants to the wild-type phenotype were obtained after screening by hygromycin B. Most transformants exhibited low aerobic mycelia, dense mycelia, transparent and curl hypha, without conidial production.The above results can summarize the function of these genes: 1) St3HNR gene was involved in melanin biosynthesis and related to penetration of appressorium, pathogenicity, respectively. 2) This study presents evidence that S. turcica possesses functional laccase and at least two laccases genes, but StLAC1 gene is not essential for melanin biosynthesis. 3) Accumulation of melanin in S. turcica was affected by StMR gene and it is necessary to clarify the regulation mechanism in future. This work is helpful for us to study and understand the other conserved gene function in DHN melanin biosynthesis pathway of S. turcica.更多还原