【作者】 黄勤清；

【导师】 关雄；

【作者基本信息】 福建农林大学 ， 农药学， 2006， 博士

【摘要】 蚊虫传播着各种可怕的疾病，是人类生存和健康的大敌。为寻找高效的苏云金芽孢杆菌(Bacillus thuringiensis，简称Bt)杀蚊菌株资源，本文从国家一级风景保护区武夷山等福建省多个地区采集土壤样品，采用热处理法从125份土壤中分离出71株Bt分离株，并对致倦库蚊进行生物测定，初筛得到5株(QQ13、QQ17、QQ42、QQ66和QQ92)有效菌株，其中QQ17、QQ66和QQ92有较高的毒性。这将对致倦库蚊的生物防治起到重要的促进作用。作为主要的微生物杀虫剂，Bt在孢子囊期合成蛋白水解酶。然而，Bt蛋白酶是否对杀虫晶体蛋白的功效有影响仍然是个争议性的话题。为探讨Bt特异蛋白酶在细胞生长及晶体蛋白稳定性中的作用，以及进一步探索这些蛋白酶在工业应用上的可能性，研究设计了蛋白酶基因保守区引物，对Bt的蛋白酶基因片段进行克隆及初步分析。结果表明，从Bt库斯塔克亚种菌株8010中获得了6个蛋白酶基因片段，即中性蛋白酶A、色氨酸合成酶β链、色氨酸合成酶α链、碱性蛋白酶A、磷酸化水解酶基因和糖原磷酸化酶及1个未知功能的DNA片段(可能是编码蜡质芽孢杆菌组特有蛋白的基因)。此外，本文报道了一个非常简单而且快速的提取苏云金芽孢杆菌总DNA方法。近年来，对Bt的增效途径探索逐渐成为其研究热点之一。其中，开发新剂型是重要手段之一。而开发一种新剂型，助剂是关键因素。良好助剂可改善制剂的理化性质并提高使用效果。通过对5种助剂进行筛选，本研究提出了一个新的Bt助剂研究选择方法，得到了对该制剂既能提高湿润性又能增效的BCM-101和JFC新型助剂，以提高Bt制剂的杀虫活性。本文还研究了Bt工业发酵后处理过程，旨在探讨发酵后处理过程对成品效价的影响，优化发酵后处理过程，设计和研究Bt发酵过程的调控方案，为全程性、自动化调控发酵提供依据。生产实践证明，对于本研究的工业生产菌株而言，其最佳通气量为1：1-1：1.2，温度范围为30-33℃。此外，随着发酵的进行，发酵液的pH值也出现一系列的变化。发酵后处理过程中，喷雾干燥多用于生产高效价(16000IU／mg以上)的Bt产品，板框压滤则多用于低效价(12000IU／mg以下)产品的生产。其中以喷雾干燥为主。在喷雾干燥过程中，膜过滤及干燥时间等因素，都会间接影响产品的效价。营养型生长调节剂TA-BR主要是由生长活性物质TA和油菜素内酯BR混配而成的生物肥料，对很多种植物具有良好的增产和改善品质的功能。本文据此研制了一种新型生物药肥，它集苏云金杆菌杀虫剂与营养型生长调节剂TA-BR于一体。研究结果表明，喷施生物药肥后，质量和产量均明显提高，鲜叶亩产量增幅为6.76-26.90％，酚氨比明显降低，水浸出物、水溶性糖及维生素C均有所增加。同时，生物药肥对茶毛虫、茶尺蠖和茶蚕等茶树主要鳞翅目害虫的防效可达90％以上，且对天敌安全。综上所述，本文从菌种的分离和鉴定、毒力相关基因的获得、助剂的筛选、工业发酵过程监控与分析和生物药肥的研发及应用等方面对Bt进行研究。在第2章，本研究进一步丰富了Bt菌种资源，筛选到3株对致倦库蚊高效的Bt菌株，为高效杀蚊微生物制剂的研发奠定了良好基础。通过第3章的研究，我们利用蛋白酶基因保守区引物得到了6个蛋白酶基因片段，获得了一个可能是蜡质芽孢杆菌组特异的蛋白基因，进一步丰富了Bt毒力相关基因的研究。在第4章，本研究采用引进的农药助剂，直接在生物农药企业试验，发现了一些新的有效助剂，用于改善Bt原粉的理化性质，提高湿润性和附着率，对Bt制剂起到较好增效作用。本文在第5章中对Bt工业发酵后处理过程进行监控，提高了Bt发酵后处理的控制能力。在第6章，本着既有杀虫作用又能促进茶叶产量和质量为研制目的研制了一种生物药肥。它的成功研发可极大地促进无公害茶叶的发展，综合实现生态效益、经济效益和社会效益。由于苏云金芽孢杆菌在生物防治中扮演着重要角色，上述工作可为苏云金芽孢杆菌的进一步研发奠定良好基础。更多还原

【Abstract】 Mosquitoes could cause severe illness of human and were the enemies of human health. In order to control them, a method with heat treatment was used to isolate Bacillus thuringiensis (Bt) with high toxicity against mosquitoes from many regions of Fujian province of China, including Wuyi Mountain. 71 isolates were obtained from 125 soil samples. The results showed that five isolates (QQ13, QQ17, QQ42, QQ66 and QQ92 ) were effective against Culex quinquefasciatus, three of which (QQ17, QQ66 and QQ92) showed high mortality. They might effectively promote the biocontrol of C. quinquefasciatusDuring sporulation, Bt synthesizes proteolytic enzymes. However, whether the protease profile of a Bt is related to the efficacy of the ICPs is still a subject of controversy. In order to investigate the role of specific protease of Bt in cell growth and in crystal protein stability and explore the possibility of the proteases to be used in the industry, PCR primers specific for the conserved regions of protease genes were designed and several PCR products were cloned and analyzed. The results showed that six protease genes, neutral protease A (nprA), tryptophan synthase beta chain, tryptophan synthase alpha chain, alkaline protease A (aprA), phosphohydrolase gene and glycogen phosphorylase, with known functions are identified from Bt subsp. kurstaki 8010, respectively, were obtained. In addition, a DNA sequence with unknown function, which may be Bacillus cereus group-specific gene, was also detected. A very simple and rapid method for extracting genomic DNA from Bt was also described.Recently, the ways to increase the efficacy of Bt have been hot spots. And research and development of new pesticide adjuvants plays a key role. Good pesticide adjuvants could improve physical and chemical characteristics of pesticide and increase the efficacy of Bt products. In current study, we had investigated 5 pesticide adjuvants for their potential usage in pesticide industries. Among the adjuvants, both BCM-101 and JFC could improve the wettbility of Bt preparation and enhance its activity to Heliotis armigera. In order to increase the efficacy of Bt, a new method of adjuvant-selection was proposed.In order to explore the effects of treatments after fermentation on Bt products, optimize the treatment procedures after fermentation, and design the regulation methods of fermentation, we have collected the data of Bt industrial treatment procedures after fermentation to lay a foundation on automatic fermentation. The results showed that as far as the indusrial Bt strain was concerned, the best air and media ratio was 1:1-1:1.2 and the good fermentation temperatures were 30-33℃. The pH values of fermentation liquid varied during the fermentation. In the treatment procedures after fermentation, membrane filtration methods were often used to produce 16000IU/mg Bt products while plate-frame pressure filtration methods were frequently applied in the production of 12000IU/mg Bt products. And the former plays a key role in the procedures. Many elements during spray-drying procedures, including membrane filtration and spray-drying time, could indirectly affect the efficacy of Bt products.The quality and yield of the tea were significantly improved after using TA-BR in combination with Bt. Tea yield could increase by 6.76%～26.90% (per mu), the proportion of the amino acids and the tea polyhenols was obviously reduced, the water extracts,the soluble sugar and Vitamin C of tea were higher than that of CK. In addition, the motability of TA-BR in combination with Bt on the Euproctis pseudoconspersa, Ectropis obliqua hypulina, Andraca bipunctata, which were the main pest damaging to tea plants, could reach above 90%. It is safe for natural enemies.In summary, the isolation and identification of Bt strains and toxin-related genes, the screen of pesticide adjuvants, the monitor of fermentation procedures and the research and development of TA-BR in combination with Bt had been characterized in this studies. In chapter 2, we obtained 3 Bt isolates with high toxicity against C. quinquefasciatus. This work might lay a foundation on biopesticides products. In chapter 3, we obtained 6 DNA fragments of protease genes and a gene fragment, which might be B. cereus group specific genes, by primer pairs specific for the conserved regions of Bt protease genes. In chapter 4, in order to improve the efficacy of Bt, several pesticide adjuvants had been tested in a Bt factory to find adjuvants with good physical and chemical characteristics, better wettability and adhesion ratio. In chapter 5, we developed a product, TA-BR in combination with Bt, to improve the insecticidal efficacy and promote the yield and quality of tea. This product could greatly promote the development of unharmful tea and make benefits of zoology, economy and society true. Since Bt plays a key role in the biocontrol of pests and plant diseases, the above finding might lay an important foundation in the further work.更多还原