【作者】 张海燕；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 环境科学与工程， 2008， 博士

【摘要】 角毛壳菌(Chaetomium cupreum Ames)是一种重要的生物防治真菌,对多种植物病原真菌均有很好的防治作用。本文以角毛壳菌(C. cupreum)为研究对象,应用cDNA文库构建、表达序列标签测定(ESTs)、生物信息学分析和转基因技术,研究角毛壳菌(C. cupreum)的生物防治分子机理,为研究和开发新型角毛壳菌(C. cupreum)微生物杀菌剂奠定坚实的基础。利用分别以葡萄糖和几丁质为碳源生长的角毛壳菌(C. cupreum)菌丝体为材料,成功地构建了角毛壳菌(C. cupreum)两个不同培养条件下的cDNA文库,分别命名为菌丝体cDNA文库(简称C1文库)和菌丝体诱导cDNA文库(简称C2文库)。C1文库的滴度为0.93×106 pfu/mL,重组率为94.2%,插入片断平均长度约1.3 kb。C2文库滴度为1.02×106 pfu/mL,重组率为95.3%,插入片断平均长度约1.2 kb。对随机挑选的cDNA克隆进行ESTs测定和生物信息学分析,C1文库共获得3069条高质量ESTs序列,它们代表了1471条单一基因(unigenes),包括392条重叠群(contigs)和1079条单拷贝基因(singlets)。其中874条单一基因与国际公共非冗余蛋白质数据库蛋白同源,代表已知功能基因,597条为未知功能的新基因。GO分类表明已知基因中与生物学过程(61.75%)相关的功能基因最多,其次是分子生物学功能基因(33.65%)和细胞组分(4.60%)功能基因。C2文库共获得1221条ESTs序列,拼接为828条单一基因,包括176条重叠群,652条单拷贝基因;468条为已知功能基因,360条为未知功能新基因。已知基因中也是与生物学过程相关的功能基因所占比例最高(63.31%),其次是分子生物学功能基因(33.77%)和细胞组分功能基因(2.92%)。C1和C2文库所获得的ESTs序列登录在国际GenBank数据库中,序列接受号为DV544375-DV548659。对C1和C2文库进行了基因表达差异分析。在两个文库中共同表达的基因是333条,C1文库中特异表达基因是1138条,C2文库中特异表达基因是495条,其中包括11条与生物防治相关的基因。对C1和C2文库进行了代谢途径差异分析。共发现角毛壳菌(C. cupreum)70种代谢途径,其中C1文库中包括67种代谢,C2文库中包括63种代谢。分析表明,角毛壳菌(C. cupreum)在与植物病原真菌斗争过程中,通过启动糖异生作用等代谢来提高自身的生长速度,以争取更多的营养和生存空间与植物病原真菌竞争。C1文库中得到20条与生物防治相关的基因,C2文库中得到23条与生物防治相关的基因,研究发现,角毛壳菌(C. cupreum)的生物防治作用是多机制协同作用的复杂体系,主要涉及细胞壁降解作用、蛋白酶水解作用、拮抗作用等。两个文库获得的生物防治基因不同。C2文库中独有的与生物防治相关表达基因主要是PTR2转运蛋白(DV547977)、β-N-乙酰葡糖胺糖苷酶(DV545166)、α-葡萄糖苷酶(DV548040)、ABC转运蛋白(DV546007)。克隆了角毛壳菌(C. cupreum)46kDa内切几丁质酶基因(Chi46,序列登录号EUI42805),利用构建的pYES2-Chi46酵母表达载体,将Chi46基因成功转化到酿酒酵母(Saccharomyces cerevisiae)H158菌株。对转化子发酵条件进行优化,结果表明:当半乳糖诱导浓度为2.5%、几丁质浓度为0.4%、诱导培养基的起始pH值为6时,可较大幅度地提高转化子几丁质酶酶活,最高可达1.96U。更多还原

【Abstract】 Chaetomium cupreum Ames is an important biocontrol fungus with effective control ability to some plant pathogenic fungi. The molecular biocontrol mechanisms were studied with the cDNA library construction, expressed sequence tags, bioinformatics analysis and transgenic technology to C. cupreum. It’s significant greatly for the research and exploitation of new type microbial-biofungicide of C. cupreum.Two cDNA libraries of C. cupreum were constructed successfully with the mycelia grown on culture using glucose or chitin as carbon source. They were named mycelial cDNA library (abbreviation: C1 cDNA library) and induced mycelial cDNA library (abbreviation: C2 cDNA library), respectively. The titer and recombinant rate of C1 cDNA library was 0.93×106 pfu/mL and 94.2 %; and the average insert size was 1.3 kb. The titer and recombinant rate of C2 cDNA library was 1.02×106 pfu/mL and 95.3 %, the average length of cDNA inserts was 1.2 kb.ESTs sequencing and bioinformatics analysis were done to randomly selected cDNA clones. 3069 ESTs of high quality in C1 cDNA library were acquired which were assembled into 1471 unigenes including 392 contigs and 1079 singlets. Among them, 874 unigenes exhibited strong similarity to proteins in non-redundancy public databases representing known genes, the others were new genes with unknown function. The results of GO ontology showed that known genes with biological process function (61.75%) accounted for most represented level, then cellular component (4.60%) and molecular function (33.65%).1221 ESTs from C2 cDNA library were acquired which were assembled into 828 unigenes including 176 contigs and 652 singlets. 468 unigenes represented known genes; the others were new genes with unknown function. Genes with biological process function had the largest scale accounting for 63.31%, genes with molecular function and cellular component function accounted for 33.77% and 2.92%. The ESTs of two cDNA libraries of C. cupreum have been deposited in the GenBank database with following accession Nos. DV544375-DV548659. The analysis of genes different expression was done to C1 and C2 cDNA library. Limited inter-library overlap of gene expression (333) was seen and 1138 unigenes were library C1 specific and 495 unigenes were library C2 specific containing 11 unigenes with biocontrol function.The analysis of metabolic pathways enabled the identification of 70 metabolism including 67 in C1 and 63 in C2 cDNA library. The results demomstrated that C. cupreum may be induced metabolism of gluconeogenesis to maintain fast cell growth rate in response to the competition provided by the plant fungal pathogen.Twenty genes and 23 genes related to biocontrol were obtained in C1 and C2 cDNA library. The analysis revealed that the biocontrol function of C. cupreum was complicated which was involved in many mechanisms including the degradation of cell wall, hydrolyzation of proteases, antagonism and so on.The biocontrol genes in two cDNA libraries were different. The special genes related to biocontrol in C2 cDNA library were PTR2 protein (DV547977),β-N-acetylglucosaminidase (DV545166),α-glucosidase (DV548040) and ABC transporter (DV546007).The full-length cDNA, encoding 46kDa endochitinase (Chi46,EU142805) of C. cupreum was successfully cloned, and transformed into Saccharomyces cerevisiae H158 using the constructed vector of pYES2-Chi46. Optimal culture condition was investigated to transformant. Ferment optimization of transformant showed that high level of chitinase activity was induced to 1.96U in the medium containing 2.5 %β-galactose, 0.4 % chitinase, pH6.更多还原