【作者】 黄彩红；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 环境工程， 2008， 博士

【摘要】 丝状土壤真菌哈茨木霉(Trichoderma harzianum)作为一种优秀的生防因子具有广泛的作用谱,在生物防治和环境保护方面发挥着重要作用。为了从基因组水平上探讨其生防机理并挖掘生防相关新基因,本实验室以胶态几丁质为唯一碳源,构建哈茨木霉(T. harzianum)菌丝体时期诱导表达的cDNA文库并获得1,386条EST序列(序列号:DY761370~DY762921),通过生物信息学分析成功获得了2条新基因(几丁质酶基因Chit37和噻唑生物合成酶基因Thi4 ), GenBank序列接受号分别为DQ647957和DQ647956。Chit37基因的氨基酸序列含有几丁质酶18家族保守活性位点“FDGIDIDIE”,该基因家族因具有降解几丁质活性而在植物病害生物防治、海洋垃圾降解及资源的再生利用过程中发挥着重要作用,全新碱性几丁质酶基因Chit37的开发和研究将丰富基因资源,为高效工程菌的构建提供重要材料。Chit37基因原核表达优化条件为0.1mmol/L IPTG诱导培养8h,这一结果对于后续研究及大规模发酵具有重要的指导意义。通过穿梭质粒pYES2实现基因Chit37在酿酒酵母(S. cerevisiae)H158中的外源表达,考虑到非翻译区域对表达产物的可能影响,使用两种目的片段Chit37-I(包含非翻译区的全长序列)和Chit37-II(只含有开放读码框序列);半定量RT-PCR结果表明,Chit37-I在诱导后96h和108h表达量较高,而Chit37-II则在诱导后84h和96h mRNA转录水平较高。DNS法检测转化子发酵周期,当接种比例为1:100时CHIT37-I和CHIT37-II转化子的产酶高峰分别为第108h和96h,最适反应温度、pH值及底物浓度均为40℃、5.0及5%,CHIT37-II具有发酵周期短、酶活性高且对底物浓度适应范围广等特点而在大规模发酵生产中更具优势,对于非翻译区作用的探讨将有助于今后对Chit37基因的合理利用。构建表达载体pPKTCP,利用根癌农杆菌介导法转入生防真菌哈茨木霉(T. harzianum)中,分子生物学检测表明,Chit37基因以单拷贝形式整合到哈茨木霉基因组DNA中且都能够稳定遗传,转化子对病原菌的抑制率显著高于原始菌株,对六种病原菌的拮抗能力由强至弱分别是核盘菌(S. sclerotiorum)、立枯丝核菌(R. solani)、尖镰孢菌(F. oxysporum)、齐整小核菌(S. rolfsii)、茄腐镰刀菌(F. moniliforme)、和灰霉病菌(B. cinerea)。这一结果说明CHIT37具有抗真菌活性并在哈茨木霉生物防治过程中发挥着重要作用,转化子可直接以菌剂形式应用于病害防治,具有非常重要的理论意义和实际应用价值,并有助于促进生物防治的产业化进程。Thi4基因与Thi4家族具有较高的同源性,该基因家族的功能与逆境胁迫相关,作为维生素B1生物合成途径中的关键酶而间接参与有机体新陈代谢,同时噻唑作为多种农药内核被广泛应用于农业生产。因此,Thi4基因的研究对生物法大量生产维生素B1、生物合成新型超高效农药及环境可持续发展都具有十分重要的意义。基因原核表达优化条件为0.1mmol/L IPTG诱导4h;将基因构建到穿梭质粒pYES2上实现在酿酒酵母(S. cerevisiae)H158中的外源表达,Northern杂交分析表明该基因可经半乳糖诱导转录表达,酵母高效工程菌株的构建为工业化生产酶制剂提供了材料和依据。更多还原

【Abstract】 Trichoderma harzianum is a filamentous soil fungus known as an effective biocontrol agent for a wide range of economically important airborne and soilborne plant pathogens. It plays a key role in biocontrol and environmental protection. To discover biocontrol mechanism in the level of genome and explore genes associated with biocontrol, an induced cDNA library of T. harzianum T88 mycelium was constructed by using colloidal chitin as sole carbon source and 1,386 ESTs were obtained (accession no. DY761370~DY762921). A novel endochitinase gene Chit37 and a novel thiazole biosynthetic enzyme gene Thi4 were obtained successfully by this means. The sequences of two genes were published on the GenBank with the accession numbers of DQ647957 and DQ647956.The animo acids sequence of gene Chit37 shared higher sequence identities with chitinase 18 family and contained a highly conserved motif (FDGIDIDIE) which corresponded to a catalytic domain. This family hydrolyze chitin so that it palys a very important role in agricultural biocontrol, ocean wastes degradation and resources recycling. The exploitation of novel chitinase gene Chit37 will supply the valuable materials for construction of efficient engineering strains. The gene Chit37 was expressed in E. coli BL21 (DE3) and the optimal induced conditions was 0.1mmol/L IPTG, 8h. This result will be significant to the preceeding research and large scale fermentation. Heterologous expression of the gene in S. cerevisiae H158 was carried out. Northern blotting analysis demonstrated that the gene was expressed at the transcription level. Considering the possible effects of untranslated regions (UTRs), two kinds of fragments of Chit37 were used in this study, the full-length gene (Chit37-I) and the coding region of the gene (Chit37-II). Semi-quantitative RT-PCR indicated that Chit37-I exhibited high expression level on 96h and 108h after galactose induction, while Chit37-II showed high mRNA transcription level on 84h and 96h. Both of the expressed products secreted to the medium and were measured in active forms. The optimal reaction temperature, pH and substrate concentration of CHIT37-I and CHIT37-II were 40℃, 5.0 and 5 %. However, the optimal culture time was 108h and 96h respectively. The CHIT37-II showed higher activity than CHIT37-I throughout and indicated the advantages in large scale fermentation. Discussions on the action of UTRs will benefit the reasonable utilization in the future.Expression vector pPKTCP was constructed and was transformed into T. harzianum mediated by A. tumefaciens. Molecular analysis showed that the T-DNA was integrated into the genome of T. harzianum with a single insert of T-DNA. The transformants were stable through mitotic and meiotic cell divisions. There was significant difference in antifungal activities between original strain of T. harzianum and its transformants. The conidial germination and mycelial growth of S. sclerotiorum, R. solani, F. oxysporum, S. rolfsii, F. moniliforme and B. cinerea were significantly inhibited in varying degrees. This result indicated that CHIT37 had antifungal activity and played a key role in biocontrol of T.harzianum. Besides, transformants could be used as inocula against plant diseases. This work has important theoretical significance and practical application value and will promote the industrialization process of biocontrol. Gene Thi4 was found sharing higher sequence identities with Thi4 family. Its function associated with abiotic stress and involved in organismic metabolism as the key enzyme of vitamin B1 biosynthesis. In addition, it was widely used in agricultural production as the framework of many pesticide and herbicide thiazole. Therefore, its molecular cloing and analysis have important significant for mass production of vitamin B1, super-effective pesticides by biological method and environmental sustainable development. The gene Thi4 was expressed in E. coli BL21 (DE3) and the optimal induced conditions was 0.1mmol/L IPTG, 4h. Heterologous expression of the gene in S. cerevisiae H158 was carried out. Northern blotting analysis demonstrated that the gene was expressed at the transcription level. The construction of efficient engineering strain supplied materials and basis for the industrial production of enzyme preparation.更多还原