【作者】 咸洪泉；

【导师】 李多川；

【作者基本信息】 山东农业大学 ， 经济微生物学， 2011， 博士

【摘要】 菌寄生真菌产生的几丁质酶直接参与寄生过程,可降解病原真菌的细胞壁,抑制病原真菌的菌丝生长、孢子萌发和芽管伸长,普遍认为是一种与抗真菌病害有关的酶。菌寄生真菌几丁质酶的应用非常广,除了用于植物病害的生物防治之外;还可用于几丁质的生物转化,几丁质经几丁质酶部分水解后产生生物活性物质几丁寡糖,被广泛应用在农业、医药、化工、食品、环境等许多领域。转真菌几丁质酶基因的作物具有高的抗病性,抗病性持久,抗病谱广,而且还可以提高抗虫能力。目前,菌寄生菌几丁质酶及其基因研究主要以木霉菌为主,对其他菌寄生真菌几丁质酶基因的克隆和转化研究较少。因此从其他菌寄生真菌资源中克隆新的几丁质酶基因,研究其表达产物的酶学性质和抑菌抗病活性,对于生物防治和植物分子抗病育种研究具有重要意义。粉红聚端孢（Trichothecium roseum）和黄蓝状菌（Talaromyces flavus）是两种重要的菌寄生菌,本研究运用RT-PCR、RACE-PCR和TAIL-PCR技术,从中克隆了3个完整的几丁质酶基因和1个几丁质酶基因的部分序列,成功的在毕赤酵母中表达了3个几丁质酶基因,研究了表达产物的性质;将克隆自T. roseum的几丁质酶基因转入烟草,检测了转基因植物的抗病性。1利用几丁质酶同源保守序列设计简并引物,运用RT-PCR、RACE-PCR和TAIL-PCR技术,从菌寄生菌T. roseum中克隆一个几丁质酶基因trchi1。该基因包含一个1278 bp的开放阅读框ORF,编码425个氨基酸,具有由20个氨基酸组成的信号肽序列,属于糖苷水解酶18家族几丁质酶。该几丁质酶基因mRNA在GenBank中的登录号为GU361768,DNA登录号为GU361767。构建酵母表达载体将trchi1基因转化到毕赤酵母中进行分泌表达,得到了有几丁质酶活性的目的蛋白,该蛋白分子量为43.97 kDa,最适反应温度为50℃,最适反应pH值为6.5,45℃以下、pH 6-8之间稳定;金属离子Hg²⁺、Cu²⁺、Ag+、Zn²⁺、Fe²⁺对表达的几丁质酶有显著的抑制作用,最适底物为胶体几丁质。重组几丁质酶对板栗疫病病菌菌丝生长抑制作用较强,可强烈抑制烟草炭疽病菌和烟草赤星病菌的孢子萌发;对玉米弯孢病菌的孢子萌发有明显的影响,在低浓度下可促进孢子萌发,但是在高浓度下能抑制孢子萌发。2从菌寄生菌黄蓝状菌（Talaromyces flavus） ACCC30960菌株中克隆了tfchi1和tfchi2 2个几丁质酶基因。tfchi1基因包含一个1194 bp的开放阅读框ORF,编码397个氨基酸,没有信号肽序列,属于糖苷水解酶18家族几丁质酶。该基因mRNA在GenBank中的登录号为GU361770,DNA登录号为GU361769。tfchi1基因转化到毕赤酵母中,分泌表达了有几丁质酶活性的目的蛋白,该蛋白分子量为42.53 kDa,Tfchi1几丁质酶的最适反应温度为35℃,最适反应pH值为5.5,30℃以下、pH 6-8之间稳定;Hg²⁺、Zn²⁺、Cu²⁺、Fe²⁺、Ag⁺对几丁质酶有显著的抑制作用,最适底物为胶体几丁质。重组几丁质酶对板栗疫病病菌和杨树腐烂病菌菌丝生长抑制作用最强,对烟草赤星病菌和小麦赤霉病菌抑制作用较弱,酶浓度越高抑制作用越强;几丁质酶对玉米弯孢病菌的孢子萌发有明显的抑制作用,并且孢子萌发后芽管基本不再伸长。tfchi2基因包含1个1191 bp的开放阅读框ORF,编码396个氨基酸,没有信号肽序列,属于糖苷水解酶18家族。该基因在GenBank中的登录号为HQ896839。构建tfchi2酵母分泌型表达载体并转化到毕赤酵母中,成功表达了有几丁质酶活性的目的蛋白,该蛋白分子量为44.06 kDa,Tfchi2几丁质酶的最适反应温度为70℃,最适反应pH值为6,在55℃以下、pH 5-9之间稳定;Ba²⁺、K⁺对重组几丁质酶有激活作用,Fe²⁺、Hg²⁺、Mg²⁺可完全抑制几丁质酶活性,Cu²⁺、Zn²⁺、Ag⁺、NH₄⁺等对表达的几丁质酶有显著的抑制作用,特异性降解胶体几丁质。重组几丁质酶可抑制病原真菌菌丝生长、孢子萌发和芽管伸长。其中对板栗疫病病菌和杨树腐烂病菌菌丝生长抑制作用较强,在低浓度下可抑制烟草炭疽病菌的孢子萌发,在高浓度下可完全抑制烟草炭疽病菌的孢子萌发;在较低浓度下可促进烟草赤星病菌和玉米弯孢叶斑病菌孢子萌发,在高浓度下可抑制孢子萌发,并且可抑制萌发后芽管的伸长。3运用RT-PCR、RACE-PCR技术,从T. flavus菌株ACCC30404中克隆了1个几丁质酶基因tfchit3的部分序列,已克隆的tfchit3基因序列cDNA长865 bp,终止密码子TAA,编码267个氨基酸。属于糖苷水解酶18家族。4构建了几丁质酶trchi1的植物表达载体,通过农杆菌介导的叶盘法成功地转化了烟草,Southern杂交及表达分析试验结果表明,trchi1基因已经整合到烟草基因组中,并在烟草中有效表达。转几丁质酶基因trchi1的烟草对烟草赤星病和炭疽病抗性明显增强。更多还原

【Abstract】 Chitinases produced by mycoparasites are directly involved in the parasitic process. Chintinase is thought to be an enzyme related to fungi disease resistance, because it can degrade the cell wall of pathogenic fungal hosts, suppress mycelium growth, spore germination and germ tube elongation. Except in bio-control against plant diseases, mycoparasitic chitinase is widely used in bio-conversion of chitin, where chitin can be partially hydrolyzed by chitinase to generate chitooligosaccharide. Chitooligosaccharide is a kind of bio-activator with broad application in agriculture, medicine, chemical, food industry and environmental fields. Transgenic crops encoding mycoparasitic chintinase gene have high disease resistance with long persistence and broad spectrum and their pest resistance could also be enhanced. So far, studies in chitinase and its genes are mainly focusing on Trichoderma spp., but less on other mycoparasites. Cloning chintinase genes from other mycoparasites will lead us to understanding its enzymatic properties and activities to suppress pathogens, which is important for researches in bio-control and breeding disease-resistant crops at the molecular level.Trichothecium roseum and Talaromyces flavus are two important mycoparasites. In this work, three chitinase genes were completely and one was partially sequenced under using RT-PCR, RACE-PCR and TAIL-PCR technology. These three chitinases with complete sequences were successfully expressed in Pichia pastoris and their properties were well studied in this work. One of the chintinases from T. roseum was transformed into tobacco and its effect on disease resistance was analyzed.1 Degenerate primers were designed according to conserved regions in homologous sequences of chitinase genes. Chitinase gene trchi1 （accession number in GenBank GU361768 for mRNA, and GU361767 for DNA, respectively） was cloned from T. roseum, by using RT-PCR, RACE-PCR and TAIL-PCR technology. trchi1 belonged to Family 18 glycoside hydrolases, its ORF was 1278 bp and it encoded 425 amino acids containing a signal peptide with 20 amino acids. Yeast expression vector with trchi1 was transformed into Pichia pastoris to produce secretory protein. A protein, Trchi1, its molecular weight was 43.97 kDa, with optimal temperature was 50℃and optimal pH 6.5. Trchi1 also was stable at the temperature below 45℃and pH ranging from 6-8. Its activity was significantly suppressed by metal ions such as Hg²⁺, Cu²⁺, Ag⁺, Zn²⁺ and Fe²⁺. Its optimal substrate was colloidal chitin. It could strongly inhibit mycelium growth of Cryphonectria parasitica, and could obviously suppress spore germination of Colletotrichum nicotianae and Alternaria alternate, it had significant effect on spore germination of Curvularia lunata, namely promoting germination at low concentration, but inhibiting at high concentration.2 Two chitinase genes, tfchi1 and tfchi2, were cloned from T. flavus ACCC3096. tfchi1 （accession number in GenBank GU361770 for mRNA, and GU361769 for DNA, respectively） was 1194 bp and encoded 397 amino acids which had no signal peptide. The chitinase encoded by tfchi1 was a member of Family 18 glycoside hydrolases. A protein, Tfchi1, having chitinase activity was expressed in Pichia pastoris. Its MW was 42.53 kDa, optimal temperature at 35℃and optimal pH at 5.5. Tfchi1 was stable at the temperature below 30℃and ranging from 6-8. It was significantly suppressed by Hg²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and Ag⁺. Its optimal substrate was colloidal chitin. It had strongest inhibition to mycelium growth of Cryphonectria parasitica and Cytospora chrysosperma, but had weaker inhibition to that of Alternaria alternate and Fusarium graminearum. The higher chitinase concentration there is, the stronger inhibition is observed. It significantly influenced spore germination of Curvularia lunata and the germ tube hardly extended after the spore germination.The gene, tfchi2 （accession number in GenBank was HQ896839） included an ORF with 1191 bp and encoded 396 amino acid which had no signal peptide. The chitinase encoded by tfchi2 was also involved in Family 18 glycoside hydrolases. A protein, Tfchi2, with chitinase activity was detected. Its MW was 44.06 kDa, with optimal temperature at 70℃and optimal pH at 6. Tfchi2 was stable at the temperature below 55℃and pH ranging from 6-9. It could be activated by Ba²⁺, K⁺, completely inactived by Fe²⁺, Hg²⁺, Mg²⁺ and significantly reduced its activity by Cu²⁺, Zn²⁺, Ag⁺ and NH4. The protein had a specific activity to degrade colloidal chitin. It inhibited mycelium growth of fungal pathogen, as well as spore germination and germ tube extending. Therein, strong inhibition to mycelium growth of Cryphonectria parasitica and Cytospora chrysosperma was observed. It partially inhibited spore germination of Colletotrichum gloeosporioedes at lower concentration, while completely inhibited it at high concentration. But results also showed that spore germination of Alternaria alternate and Curvularia lunata was facilitated at low recombinant chitinase concentration but inhibited at high concentration. High concentration of recombinant chitinase also inhibited germ tube elongation after spore germinating.3 One partial sequences of chitinase gene, tfchi3 were cloned from T. flavus ACCC30404. cDNA of the cloned sequences had 865 bp with TAA as termination codon and encoded 267 amino acid which was predicted as a member of Family 18 glycoside hydrolases.4 Plant expression vector with gene trchi1 was constructed and successfully transformed into tobacco using Leaf Disc via Agrobacterium rhizogines. The Southern blot and protein expression analysis result indicated that trchi1 was integrated into tobacco genome, and was expressed effectively in tobacco. It was observed that the resistance of tobacco with gene trchi1 against Alternaria alternate and colletotrichum gloeosporioedes was obviously enhanced.更多还原