【作者】 Abebe Jenberie Wubie；

【导师】 周婷；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2014， 博士

【摘要】 蜜蜂是世界上最重要的经济昆虫,在世界上只要有花和花粉的地方就有蜜蜂。然而在生态上如此重要的昆虫却遭受着各种各样疾病的侵染,这些疾病可能由多种病原物引起,如真菌就是其中一种重要的病原微生物。对真菌病害的致病性机理的研究一直以来都是研究的热点。蜜蜂白垩病是由蜜蜂球囊菌(Ascosphaera apis)引起的,主要侵染蜜蜂的幼虫。研究表明,白垩病可致使蜜蜂蜂蜜产量减产5%-37%,使蜜蜂幼虫产生80%的死亡率。近些年来,由于蜜蜂白垩病发病较重,人们对杀菌剂使用较乱,导致蜂产品污染严重。再加上蜜蜂的转地饲养,蜜蜂长距离的运输又导致了蜜蜂白垩病的传播具有全球性。更为严重的是,引起蜜蜂白垩病的致病菌球囊菌的分生孢子可以在蜂蜜、花粉、蜂机具及发病蜂场的土壤中存活15年以上,当条件适宜其生长时,分生孢子会从环境中萌发出来,再次传播,使得该病很难根除。尽管科研人员提出了许多防控蜜蜂白垩病的技术措施,然而至目前为止,对蜜蜂白垩病的防治还是非常困难的。鉴于此,本研究采用分子生物学方法,利用限制性内切酶介导的基因整合技术构建了蜜蜂球囊菌遗传转化体系,获得了球囊菌致病性降低突变体,对蜜蜂球囊菌致病的分子机理进行了初步的探讨。1.蜜蜂球囊菌原生质体制备及其再生构建蜜蜂球囊菌REMI转化体系首先要制备出产量高再生率高的原生质体。本实验对影响蜜蜂球囊菌菌株原生质体制备及再生的因子进行了系统的研究,同时对蜜蜂球囊菌原生质体的释放及再生过程进行了显微观察。结果表明,采用液体培养基进行24hrs培养的蜜蜂球囊菌菌体,在28℃条件下,应用50mg/mL的崩溃酶,经4hrs酶解所制备的原生质体释放量最大,达到98.36×105/mL。在上述条件下,采用0.8mol/L柠檬酸与NaCl的混合液作为稳渗剂,原生质体的再生率也较高,达到51.06%。蜜蜂球囊菌以菌丝断裂方式释放原生质体,原生质体再生时表现为原生质体先是突出,其后延长,并最终发育成为正常菌丝。2. REMI转化子的获得及其验证采用REMI方法获得了11个转化子。经过PCR. ClustalW多序列比对及Southern blot方法验证,所获得突变体含有目的基因的插入,由此证明上述转化子为蜜蜂球囊菌突变菌株。3.蜜蜂球囊菌REMI突变子致病性鉴定通过幼虫接种实验,对所获得的REMI突变菌株进行了致病力进行了检测。结果表明,所获得的突变体菌株同原始菌株比较,致病力差异显著(p<0.01),接种后的第5天野生型菌株引起幼虫发病率达19.9%,病情指数达33.5,M4和M8的致病性明显小于野生型菌株,幼虫发病率仅为16.8%和14.3%,病情指数为28.9和23.7。同时,突变体菌株在侵染蜜蜂幼虫后,蜜蜂发病过程及幼虫死亡率均较野生型有较大差异。更多还原

【Abstract】 Honeybees are one of the most well-known economically beneficial social insects which can be kept almost anywhere in the globe as long as there are flowering plants that produce nectar and pollen. These insects are accomplishing about80%of all crop insect pollinations. However, this ecologically important social insect is suffering from various diseases caused by various pathogens, like fungal diseases. Of course, fungi are economically important organisms whose pathogenicity continues to be the focus of extensive research. Among the fungal diseases, chalkbrood is the one known to be an invasive mycosis in honeybees, caused by Ascosphaera apis, that exclusively affects honeybee brood. The disease has been reported to cause about5%-37%reduction in honey production and80%brood death. Consequently, the severity of the disease, these days, has increased the use of pesticides and frequent long-distance transportation of colonies which is believed to provide an additional opportunity for A. apis infection globally. Furthermore, the fact that A. apis is so widespread in the globe and its viable spores can be found in stored honey, pollen, pollen capsules/tablets, used hive components, used beekeeping tools and equipments, and possibly in soil around infected apiaries for more than15years, makes the possibility of its eradication most unlikely. Here, even though a broad range of experimental works have been conducted safe control of the disease has been noticed to be difficult. Thus, genetic engineering works for better manipulation of the fungus, molecular investigation of pathogenesis and possible identification of strategies to control the disease shall take current issue of research. With this, this work was conducted with the objectives of understanding and developing an efficient and reproducible technique for the ultimate purpose of making this fungal species amenable to genetic studies and transformation. Furthermore, this work was aimed to study the pathogenicity of restricted enzyme mediated integration engineered A. apis mutants in honeybee(Apis melifera L.) larvae. Consequently, as restricted enzyme-mediated integration enables the investigation of the infectivity of transformed pathogens to host organisms, this work has utilized protoplast isolation and transformation techniques as a tool to understand this fungus further to meet set objectives. According to the results obtained, the fungus showed varying responses in terms of yield, size, and regeneration rates on different mediums, and osmotic stabilizer combinations. In this way, it was confirmed that younger incubation time has yielded better mycelium suitable for highest protoplast isolation while use of driselase was the best enzyme treatment, yielded about98.36×105mL-1of protoplasts. Young aged and exponentially growing mycelial culture provided the highest protoplast formation with higher protoplast viability. Use of citric acid monohydrate with NaCl as an osmotic stabilizer supported a51.06%viable protoplasts to regenerate and able to form mycelia colonies, while a combination of4hrs enzymolysis time with this osmotic stabilizer have supported53.06%protoplast regeneration. Following the successful protoplast isolation and restricted enzyme-mediated integration, PCR, ClustalW multiple sequence alignment, and Southern blot analyses confirmed the successful integration of a foreign DNA, as an insert, to the host chromosome targeting to cause mutation. An in-vitro bioassay experiment included in this work has confirmed that all engineered mutants had notable differences in pathogenicity among themselves and with the wild strain to the honeybee larvae (p<0.01). Disease infectivity data confirmed that the original (wild type) fungus caused the maximum (19.9%) amount of larval death starting from the5th day post inoculation, and its disease index was recorded to be33.5. Even though the mortality of engineered mutants was not significantly augmented compared to that of the wild strain, mutants4and8had16.8%and14.3%larval death with28.9and23.7of disease index, respectively. More specifically, transformed A. apis mutants were relatively less pathogenic to in vitro-reared honeybee larvae than those of the wild-type. In addition, higher larval death and faster mummification was detected in the wild-type too. Thus, the pathogenicity differences among strains, even in the natural conditions, have magnified the significance of further investigations on the reasons for variations among transformants and their wild types. Even if we have studied this fungus for a while, control of this fungus is still a mystery. Thus, we, generally, recommend that further understanding on this fungus and different investigations shall be carried out as a part of future important activities.更多还原