【作者】 胡宗利；

【导师】 蔡绍晳； 夏玉先；

【作者基本信息】 重庆大学 ， 生物医学工程， 2005， 博士

【摘要】 蝗虫是世界性的害虫,给农业生产带来了严重的经济损失。金龟子绿僵菌是一种蝗虫病原真菌,在蝗虫生物防治中起着重要作用。目前,世界上已经有十多个金龟子绿僵菌菌株登记注册,并实现了商品化,但这些产品常常不稳定。阻碍绿僵菌杀虫剂大规模应用的主要原因之一是孢子的储藏和环境稳定性问题。本实验室从我国蝗虫病原菌中分离、选育出一株高毒力、高产孢、强专化性的杀蝗新菌株金龟子绿僵菌CQMa102,用该绿僵菌生产的孢子粉及其油悬浮剂已在国内登记注册,正逐步实现商品化,却也同样存在进一步延长储藏期,增强环境稳定性的问题。大量研究报道,海藻糖是广泛存在于各种生物体内的抗逆境剂,具有增强生物体对高温、脱水、干旱、冷冻、高渗透性、重金属及有毒试剂等逆境的抵抗能力。胞内海藻糖积累与丝状真菌孢子的储藏期延长密切相关。将酿酒酵母细胞内中性海藻糖酶基因敲除后,中性海藻糖酶活性丧失,海藻糖含量提高3 倍。由此我们可以推测,通过基因工程技术改变绿僵菌体内海藻糖代谢途径,可以提高孢子内海藻糖含量,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。要通过改变海藻糖代谢途径来改良绿僵菌菌种,就必须了解绿僵菌体内海藻糖的代谢和调控,但是人们对绿僵菌体内海藻糖的代谢研究得较少,夏玉先等首先克隆了金龟子绿僵菌中性海藻糖酶基因,但其生物学功能还未见报道。本研究拟通过增加金龟子绿僵菌中性海藻糖酶基因的拷贝数以减少孢子形成过程中海藻糖的合成,采用基因破坏技术去除或降低金龟子绿僵菌中性海藻糖酶活性以增加孢子中海藻糖含量,并进一步分析孢子中海藻糖的含量及其与孢子抗逆能力和耐储藏能力的关系,从而阐明中性海藻糖酶在孢子耐热力和储藏稳定性中的作用。本研究利用PCR、RT-PCR、RACE、panhandle PCR 等方法成功地克隆了金龟子绿僵菌CQMa102 中性海藻糖酶基因,并登录NCBI 的GenBank, 登录号为:AY557613（基因组序列）, AY557612（cDNA 序列）。根据该基因序列构建了金龟子绿僵菌中性海藻糖酶超表达载体、同源置换载体和RNA 干扰载体,分别利用PEG-CaCl₂ 介导法、电转化法和基因枪法,转化金龟子绿僵菌CQMa102,经过PCR 筛选和Southern blot 验证,获得3 个中性海藻糖酶超表达转化菌和3 个RNAi 转化菌。利用Northern blot 分析这几个突变菌株中性海藻糖酶基因mRNA 的含量,选择中性海藻糖酶基因超表达2 倍和中性海藻糖酶基因被抑制到42%的两个转化菌:Ma113 和Ma688,将其与出发菌株进行分生孢子内海藻糖含量的测定,孢子储藏更多还原

【Abstract】 The locust is a cosmopolitan pest and has brought serious economic loss to agriculture. Metarhizium anisopliae, an entomopathogenic fungus widely applied in domestic and abroad, has an important role in biological control of locust. At present, many strains of M. anisopliae have been registered in the world and have realized commerciality. However, these products usually show instability. The storage and environmental instability of conidiospores is a significant drawback of commercial scale use of this very promising alternative to chemical pesticides. We have isolated a strain of M. anisopliae var acridum CQMa102 exhibiting a high degree of locust specificity from natural environment, and registered its conidial powder and oil suspending preparation in China, and used it on a commercial scale. Whereas, there still has the problem of prolonging storage time and enhancing environmental stability. Trehalose, a non-reducing disaccharide, occurs in a large range of organisms, such as bacteria, fungi, animals and plants. In addition to its function as a storage carbohydrate and transport sugar, trehalose plays an important role in stress protection, especially during heat stress, dehydration, drought, freeze, high saturation, heavy metal and noxious reagent. There is a strong correlation between intracellular trehalose accumulation and prolonged storage time of conidia from filamentous fungi. The activity of neutral trehalase lost and trehalose concentration has been increased three times after knocking out neutral trehalase gene in S. cerevisiae. Based on these research results, we 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 application of genetic engineering. In order to improve M. anisopliae strain by changing trehalose metabolism pathway, we must understand metabolism and regulative mechanism of trehalose in M. anisopliae. Nevertheless, Little attention has been given to the research of trehalose in M. anisopliae. A neutral trehalase gene in M. anisopliae was first cloned and characterized by Xia et al, but its biological role in M. anisopliae has still remained unclear. It seemed therefore worthwhile to get more information on the role of neutral trehalase during storage of conidiospores in entomopathogenic fungus M. anisopliae. For that purpose, in our research, we will increase the copies of neutral trehalase gene in M. anisopliae to decrease the trehalose synthesis during conidiation, and will knock out or knock down the activity of neutral trehalase to increase trehalose content in conidia by knocking out technology or double-stranded RNA interference. Furthermore, in order to elucidate the role of neutral trehalase in conidiospores stability during storage and heatshock, trehalose concentration, storage duration , thermotolerence of the mutants and wild-type strain were analyzed. In our research, the neutral trehalase gene in M. anisopliae was successfully cloned by PCR, RT-PCR, RACE and panhandle PCR, and submitted to GenBank, accession number are AY557613(genomic sequence) and AY557612(cDNA sequence). According to the sequences above, an over-expression vector and two homologous vectors and a double-stranded RNA interference vector have been constructed, and transformed to strain CQMa102 by PEG-CaCl2 mediated method, electroporation and particle bombardment. After PCR selection and Southern blot verification, three over-expression transformants and three RNA interference transformants have been obtained. Analysis of these mutants revealed that the expression level of the neutral trehalase gene in RNAi mutant (Ma688 strain) was reduced to 42% of which found in wild-type strain and the over-expression mutant (Ma113 strain) showed approximatlely 2-fold higher levels of neutral trehalase mRNA than that found in wild-type control. Then the transformants Ma688 and Ma113 were chosen for closer examination, including trehalose content, storage longevity , thermotolerance and growth characteristics. The main results were as follows: ⑴During the maturation of conidiospores, the trehalose content increased rapidly in M. anisopliae. The wild-type strain accumulated trehalose more slowly than the RNAi mutant, and more quickly than the over-expression mutant. ⑵During the accelerating aged storage, the storage duration of mature conidiospores is higher than immature ones. The storage duration of wild-type conidia is lower than that of RNAi mutant conidia, and higher than over-expression mutant ones. ⑶The ability to heatshock of mature conidia is higher than that of immature ones at 45℃. The wild-type conidia show lower thermotolerence ability than RNAi mutant conidia, and higher thermotolerence ability than over-expression ones. ⑷No significant differences in growth condition and sporulation frequencies between the wild-type strain and trehalase mutants (RNAi mutant and over-expression mutant) were observed. ⑸Based on above results, we can conclude that the biological function of neutraltrehalase in M. anisopliae assists in control of trehalose concentration via degradation of trehalose, and indirectly influences the stability of conidia during storage and during heat stress. This research achievement could provide a new basis and technology reserve for improving entomopathogenic fungus strains to have longer shelflife and strong resistance to adverse conditions by ferment technique and breeding technique including genetic engineering.更多还原