【作者】 茅洁瑜；

【导师】 林毅； 张杰；

【作者基本信息】 华侨大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 鞘翅目害虫常年给农、林业生产带来重大损失,目前采取的主要防治措施是使用化学杀虫剂,但是化学杀虫剂的弊端日益突出。一方面化学杀虫剂对生态系统影响大、药物残留高、对人畜安全性低,另一方面部分害虫已经对其产生抗性。为了减少污染、提高防治效率,国内外都在探索生物防治这一新的防治技术和方法。苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是目前研究应用最广的一类微生物杀虫剂。Bt的杀虫活性与其携带的编码杀虫晶体蛋白(Insecticidal Crystal Proteins,ICPs)的cry基因密切相关,寻找新的高毒力菌株及基因一直是研究的热点。本文应用PCR-RFLP和生物信息学两种方法开展对鞘翅目有活性的新cry基因的挖掘工作,并对所寻找到的新基因进行了表达研究,主要结果如下:1.以实验室分离的500株苏云金芽胞杆菌菌株为材料,通过杀虫活性测定筛选到43株对鞘翅目金龟科幼虫(蛴螬)有活性的菌株,其中菌株C9对三种供试昆虫均有活性。形态学观察发现这些菌株均释放球形晶体,SDS-PAGE分析显示它们均表达约130 kDa蛋白。采用PCR-RFLP法对这43株菌株进行了cry基因类型鉴定,结果显示:其中21株只含cry8Ca;13株同时含cry8Ca和cry8Ea基因;菌株QCM的PCR-RFLP图谱不同于已发现的cry8基因,说明其中含有新基因。根据已知的cry8基因全长序列设计引物,成功克隆了该新基因,由Btδ-内毒素基因国际命名委员会正式命名为cry8Xxx1。2.从已进行基因组测序的菌株NARC Bt17和HD868中,通过生物信息学方法筛选到3个对鞘翅目害虫有潜在毒力的新cry基因。成功克隆了上述基因,并命名为cry61Xxx、cry7Xxx和cry8Ma3。3.以大肠杆菌Rosetta (DE3)为宿主菌,利用表达载体pEB对所克隆的4种新cry基因进行表达,经SDS-PAGE检测,均成功表达130 kDa左右的蛋白。4.通过穿梭载体pSTK将cry8Xxx基因导入Bt无晶体突变株HD73-中,获得工程菌HD8X。SDS-PAGE分析表明cry8Xxx基因在其中能正常表达,并形成球形晶体。综上所述,本研究通过PCR-RFLP与生物信息学筛选的方法,克隆了4种新cry基因,构建了新型Bt工程菌HD8X,丰富了我国对鞘翅目有活性的基因资源,拓宽了cry基因挖掘的思路,并为应用奠定了基础。更多还原

【Abstract】 Coleopteran pests caused heavy losses to agriculture and forestry every year. At present the main control measures being taken is the use of chemical pesticides. However, the obvious disadvantage of chemical pesticides is increasing: they brought out negative impact on the ecosystem; the residue of toxin is high; they were not safe to human and livestock and some of the pests have engendered resistance to them. In order to reduce pollution, increase the efficiency of control, scholars at home and abroad are exploring new technique and methods based on biological control. Bacillus thuringiensis (Bt) is one of the microbial pesticides that are widely used. The insecticidal activity of Bt is closely related to its insecticidal crystal protein (ICPs) coding by cry genes. Looking for new strains and cry genes with high toxicity to pests has been a hot spot in research. This work includes finding novel cry genes which are toxic to Coleopteran pests with two different methods:PCR-RFLP and Bioinformatics, at the same time the expression of these novel genes are also studied.1. 500 strains of Bacillus thuringiensis isolated in our laboratory were investigated. 43 strains which were insecticidal activity to larvae of cockchafers (Coleopteran, Scarabaeidae) were obtained. C9 was entomocidal to all the three species of cockchafers tested. Observation results of morphology indicated that all of these Bt strains contained crystals in spherical shape. SDS-PAGE analysis indicated that all these strains could express protein with 130 kD molecular mass. The study of cry gene-types of these 43 strains was carried out by PCR-RFLP method. Only cry8Ca type gene could be found in 21 strains, while both cry8Ca and cry8Ea type gene existed in 13 strains. Strain QCM may contain a novel cry8 gene for its unique PCR-RFLP patterns. Designed primers based on known cry8 genes, and cloned this gene cry8Xxx which named by International Nomenclature Committee of Bt.2. By Bioinformatics, three novel genes have been screened from strains NARC Bt17 and HD868 whose genome have been sequenced. These genes designated as cry61Xxx, cry7Xxx and cry8Ma3 are predicted to be toxic to coleopteran pests, and they are cloned successfully.3. These 4 genes above-mentioned are expressed in E. coli Rosetta (DE3) via expression vector pEB. SDS-PAGE analysis indicated that they were expressed protein of about 130kDa molecular mass successfully.4. cry8Xxx gene was transformed into a crystalliferous Bt strain HD73- by pSTK, a shuttle vector. Then a recombined strain designated as HD8X was obtained. SDS-PAGE analysis showed that the cry8Xxx gene was expressed in HD8X which can form spherical crystals.In conclusion, four cry genes were cloned via PCR-RFLP and bioinformatics methods in this work, and a new engineered strain HD8X was constructed. It enriches the gene resources of our country and provides a new approach for cry gene cloning, also it establishes a ground for application.更多还原