【作者】 蔡泽骏；

【导师】 夏玉先；

【作者基本信息】 重庆大学 ， 生物医学工程， 2009， 博士

【摘要】 绿僵菌(Metarhizium anisopliae)是一种蝗虫病原真菌,在蝗虫生物防治中起着重要作用。作为真菌杀虫剂,除了具有直接穿透寄主体壁和持续控制等特点外,它更具有无公害、无残留、害虫不易产生抗性等优点,因而受到人们的广泛关注。目前,世界上已经有十多个商品化绿僵菌菌株登记注册,但是由于孢子的储藏和环境耐受性问题,这些产品常常不稳定,从而阻碍了绿僵菌杀虫剂的大规模应用。海藻糖是广泛存在于各种生物体内的抗逆境剂,通过对蛋白质等生物大分子及脂质双分子层的保护,增强了生物体对脱水、高温、低温、冻融、自由基、缺氧、高渗及有机溶剂等胁迫环境的抵抗能力,丝状真菌胞内海藻糖积累与孢子的储藏期延长呈正相关。由此可以推测,通过基因工程技术改进绿僵菌体内海藻糖代谢途径,可以提高孢子内海藻糖含量,从而延长绿僵菌孢子的储藏期,增强其环境稳定性,促进其商品化生产和应用。海藻糖主要生物合成途径为:以葡萄糖-6-磷酸(G-6-P)和尿嘧啶二磷酸葡萄糖( UDPG )为底物,经两步催化生成海藻糖。海藻糖-6-磷酸合成酶(trehalose-6-phosphate synthase,TPS: EC 2.4.1.15)催化UDPG和G-6-P合成为海藻糖-6-磷酸(T-6-P),随后在T-6-P磷酸酯酶(trehalose-6-phosphate phosphatase,TPP: EC 3.1.3.12)催化下,T-6-P脱去磷酸基成为海藻糖。该途径在原核细胞与真核细胞中略有差别:Escherichia coli中海藻糖由独立的两个酶(TPS和TPP)协同合成;而Saccharomyces cerevisiae中海藻糖由多酶复合体(TPS complex)合成。该复合体至少含4个亚基,包括具有TPS催化功能的TPS1亚基、具有TPP催化功能的TPS2亚基以及起调节功能的TPS3和TSL1亚基,目前发现原核细胞的TPS或真核细胞的TPS1是海藻糖合成的关键酶。大量研究证实,由于海藻糖的抗胁迫作用,如果超表达TPS/TPS1基因,将增强生物体的抗胁迫能力;若敲除TPS/TPS1则作用相反。除此之外,TPS1还展现了其它丰富的生物学功能,如影响孢子的生成及萌发、真菌的侵染能力、植物胚胎的正常发育等。TPS1还与控制进入糖酵解的糖流量及糖诱导的信号传输有关。但是,有关TPS1基因及酶的作用及机理目前还远未得到明确阐明。TPS1在抗胁迫环境中的重要角色及丰富的生物学功能引起了对其研究热潮,就绿僵菌而言为解决绿僵菌孢子的储藏和环境耐受性问题提供了新思路。然而令人遗憾的是,迄今为止没有有关绿僵菌TPS1基因及酶的报道,尽管它们对绿僵菌孢子抗胁迫作用及由此解决绿僵菌杀虫剂大规模应用问题上存在的潜在前景。本研究以此出发,以昆虫病原真菌绿僵菌为研究对象,分离、克隆绿僵菌TPS1基因,对基因序列特点进行分析,调查该基因在基因组中的拷贝数,并研究该基因在菌体不同发育时期的表达模式;同时利用毕赤酵母表达体系对绿僵菌TPS1进行异源表达,纯化重组蛋白,并对重组蛋白进行酶学特性分析。本研究不仅能为有关TPS1基因及酶研究领域提供一个新物种方面的补充,并且将为发展耐储、稳定、更有商业前景的基因工程真菌杀虫剂提供前期的基础研究工作。主要研究成果如下:(1)通过同源序列比对设计引物,利用PCR、RT-PCR、RACE等方法成功克隆了金龟子绿僵菌CQMa102 TPS1基因,并登录NCBI的GenBank,登录号为:AY954915。该基因在绿僵菌基因组以单拷贝形式存在,DNA序列含有两个内含子。cDNA序列全长1837 bp,其中包含一个1554 bp的开放阅读框、一个87 bp的5’端非编码区和一个195 bp的3’端非编码区。开放阅读框编码一个含517个氨基酸序列的蛋白。用生物信息学的方法进行分析,表明该蛋白属于葡萄糖基转移酶GTB型超家族。以同源模建法研究该TPS1蛋白空间结构,发现其具有GTB型超家族蛋白的典型结构,即具有N-和C-末端两个结构域,两个结构域之间存在着较大间隙,并且两个结构域内都存在Rossmann折叠。(2)调查了绿僵菌CQMa102 TPS1基因在该菌不同发育时期的表达情况。发现该基因在孢子时期未见表达, 1/4SDA液体培养3h(孢子萌发期)可见少量表达,随后表达持续增多,24h(对数生长早期)表达达到高峰,但48h(对数生长后期)表达减少,64h(稳定生长期)基本未见表达。(3)实现绿僵菌CQMa102 TPS1基因在毕赤酵母KM71中异源表达,并完成重组TPS1蛋白的纯化,纯化蛋白分子量约58 kDa, C-末端具有6个组氨酸标签。酶活测定证明,该重组蛋白具有TPS1的催化功能,该结果说明我们所克隆的基因其编码蛋白确为绿僵菌TPS1。此外,该表达及纯化体系将为以后的结构特性研究提供充足的酶量。(4)对重组TPS1蛋白的酶学特性进行了分析,发现重组TPS1酶活最适温度为40℃,在35℃-50℃条件下都具有较高的活性(具有60%以上酶活),这种较高的最适温度水平和宽范围的温度耐受现象,与TPS1在胁迫环境下扮演关键角色的作用是相适应的。重组TPS1最适pH为6.5,并且在pH5-7.5条件下都具有相对较稳定的酶活。其保持较高酶活的pH范围中包含了绿僵菌CQMa102的专性寄主——东亚飞蝗的血淋巴的pH值,该蛋白的这种pH稳定范围适于侵染环境下的酶活表达,即在真菌侵染昆虫的早期,与菌体大量合成海藻糖的需求相适应。我们的研究还证实了磷酸对重组TPS1的抑制效应,随着磷酸浓度的增加,重组蛋白的活性迅速下降;当磷酸浓度为2mM时酶活性丧失了54.6%,而10mM磷酸浓度下85%的酶活被抑制。对重组TPS1酶动力学特性的研究表明,对G-6-P和UDPG,重组蛋白都表现出典型的米式动力学特征。相应于G-6-P和UDPG,该蛋白的米式常数(Km)分别为3.9mM和9.6 mM。就葡萄糖基受体而言,重组TPS1表现出高度的底物特异性,除G-6-P外,重组TPS1对其它分别作为葡萄糖基受体的底物,包括果糖-6-磷酸、海藻糖,葡萄糖、果糖和蔗糖在内,均未表现出催化活性。更多还原

【Abstract】 Metarhizium anisopliae, an entomopathogenic fungus, is used as commercial biological control agent of locust. As an entomopathogenic microorganism, there are many advantages in the use of the fungi such as non-residue, non-resistance and non-resurgence except for the active penetration into the host cuticle and the sustaining control for locusts. At present, there are more than 10 strains of Metarhizium anisopliae registered for commercialization. But large scale use of the fungal bio-control agents is limited partially due to the adaptability to the circumstance and the failure of conidia retaining pathogenicity during long term storage.Trehalose, an important component in fungal spores, is a disaccharide which protects against several environmental stresses, such as heat, desiccation, salt, cold, freeze thawing, radical, hypoxia and organic solvent by its protection for protein and bimolecular lamellar lipid membrane. Moreover, there is a strong correlation between intracellular trehalose accumulation and prolonged storage time of conidia from filamentous fungi. So it can be speculated that increasing trehalose content in conidia, prolonging conidial storage time and enhancing its environmental stability may be feasible through changing trehalose metabolism pathway by the application of genetic engineering.The main pathway for trehalose biosynthesis is the condensation reaction of UDP-glucose (UDPG) and glucose-6-phosphate (G-6-P) to give trehalose-6-phosphate (T-6-P) catalyzed by T-6-P synthase (TPS: EC 2.4.1.15), which is the key enzyme for biosynthesis of the disaccharide. Subsequently T-6-P is dephosphorylated to trehalose by a specific T-6-P phosphatase (TPP: EC 3.1.3.12) (2, 6). In Escherichia coli, trehalose is synthesized by the two separate enzymes, encoded by the genes otsA and otsB, respectively. This is different from Saccharomyces cerevisiae, in which trehalose is synthesized by a multisubunit complex (designated as TPS complex). The TPS complex is composed at least by four subunits TPS1 with TPS activity; TPS2 with TPP activity, and regulatory subunits of TPS3/TSL1. TPS1/TPS plays a key role in the biosynthesis of trehalose. Based on the protection from trehalose, many researches showed that the overexpression of TPS1 gene would enhance the resistence of the host to the abiotic stresses while the disruption of the gene decreased the resistence. In addition, TPS1 has showed abundant biological funtions, such as its effects on conidiogenesis, spore germination, plant embryo development and pathogenesis of fungi. Moreover, TPS1 also involved in the control of the influx of sugar into glycosis and the sugar induced signalling. However, It should be noted that the function and its mechanism of TPS1 gene and enzyme are far from illumination by now.TPS1 inspired great insterest of researchers by its key role in the resistence on abiotic stresses and abundant biological functions. For M. anisopliae, TPS1 raised a new idea to solve the problems in the shelf time and the stress resistence to the enviroment. Unfortunately, no report is available on the TPS1 gene and the corresponding enzyme properties in the fungi so far, despite their potential importance for the stress resistance of the spore. Thus, the fungi become the focus of our research. We tried to clone the TPS1 gene in the fungi, analyze its sequence properties, investigated its copy number in the fungi`s genome and study its expression at different development stages. Futhermore, the TPS1 was heterogenously expressed in yeast Pichia pastoris expression system. The recombinant protein was purified and the properties of the purified protein was analyzed. The results attained from the studies above would not only provide more knowledge to the functions of TPS1, but also provide an essential work for the development of more storable, more stable, and more business perspective fungi pesticide by genetic engineering. The main results are as follows:1) The TPS1 gene was cloned successfully from M. anisopliae CQMa102 by PCR, RT-PCR and RACE. The cDNA and its deduced protein sequences were deposited in GenBank (accession number AY954915). The gene existed one copy in the fungi`s genome, and its DNA sequence contained two introns. The 1837-bp cDNA sequence contained an 1554-bp single open reading frame encoded a protein of 517 amino acids, an 87-bp 5’untranslated region and an 195-bp 3’untranslated region. Analysis of the amino acid sequences by computer using the NCBI database and BLAST revealed that the TPS1 belonged to glycosyltransferase_GTB_type superfamily. The three-dimensional structure of the protein was constructed by homology modeling method, and the typical structure of the proteins in GTB_type superfamily was found also in the TPS1. As GTB proteins, TPS1 of M. anisopliae also had distinct N- and C- terminal domains each containing a typical Rossmann fold. There was a cleft to separate the two domains.2) By the investigation on the TPS1 gene expression at different development stages of M. anisopliae CQMa102, it could be found that no expression could be detected in spores, a small amount of expression could be detected after incubated 3h (the spore germination) in 1/4SDA liquid and then increased continously until 24h (the early exponential growth), the expression decreased at 48h (the later exponential growth) and could hardly be detected at 64h (the stationary growth).3) M. anisopliae CQMa102 TPS1 was expressed heterogenously in Pichia pastoris KM71, and the recombinant protein was purified. The purified protein was≈58 kDa with a (His)6 tag at C-terminal. The TPS1catalytic function of the recombinant protein approved the accuracy of our work in the gene cloning. It should be noted that the expression system will provide sufficient amounts of recombinant TPS1 for future structural characterization of the protein.4) The properties of recombinant TPS1 were examined. The optimal temperature was 40°C and the protein reserved nice activity in 35-50°C(more than 60% activity). The optimal pH was about 6.5 and the the protein reserved nice activity in pH5-7.5. The high optimal temperature and the broadly active temperature and pH range should be adaptable to the key role of TPS1 in the resistence to abiotic stresses. The pH range reserved nice activity contain the pH of the fungi`s host, locust in which the pH of its hemolymph is 6.0, so the active pH range of the recombinant protein should be adaptable to the essential catalytic function at the early stage of the infection on locusts. Phosphate was confirmed its inhibition to the activity of the recombinant protein, the activity decreased rapidly as the concentrations of phosphate increased. The Km value of recombinant TPS1 for UDP-glucose and glucose-6-phosphate was 9.6 mM and 3.9 mM, respectively, and the enzyme was highly specific to glucose-6-phosphate for glucosyl acceptor,更多还原