MOSQUITO BIOCONTROL VIA CLONING THE BTI TOXIN GENE

许多人类疾病的控制都建立在控制蚊子的基础上 向量。生物防治计划已经成为可能，因为最近 苏云金芽孢杆菌变种的发现以色列(Bti)，产生一种 对蚊子幼虫有毒的蛋白质晶体。这个项目的目标是 到：1)确定晶体的哪个成分构成实际的 毒素；2)鉴定编码毒素的质粒；3)克隆毒素 基因转化细菌更适合长期灭蚊。 BTI在九种蛋白质中含有一种哺乳动物和一种蚊子毒素 纯净的晶体。目前还没有关于身份的信息 这些毒素，或者实际上它们是否是单独的毒素。9种蛋白质 将通过非变性凝胶电泳法分离并检测 幼虫毒性和红细胞溶解。如果这两种毒素是不同的，它 应该有可能获得只产生 蚊子毒素，从而消除由蚊子毒素 Bti的广泛使用。 据报道，Bti的4.5md和72md两个质粒都是 毒素基因。质粒将通过电泳法分离，洗脱， 并转化为无质粒Bti的原生质体。变形金刚将 通过杂交检测并检测毒素基因表达 免疫测定和幼虫毒性。如果毒素质粒很大，它将是 用限制性内切酶消化，然后连接，直到它 缩小到更容易管理的规模。 然后将Bti毒素质粒克隆到两个更合适的细菌中： 1)球形芽孢杆菌(Baccillussphaericus)。BTI在自然界中并不能很好地生存，而 BS也是一种蚊子病原体，其生态持久性一直很好 有记录在案。因此，Bti毒素基因将连接到PUB 110 稳定转化B.S.的质粒。希望重组人会 产生Bti毒素，维持BS的生态适宜性。2) 蓝藻Anacystis nidulans(An)。BTI对 按蚊，因为它们的幼虫是表层取食者，而 Bti毒素颗粒迅速沉降到底部。因此，Bti 将毒素基因连接到pUC 104载体上，稳定转化 一种。含有气泡的AN菌株将漂浮在表面并 对按蚊幼虫仍然有效。此外，与Bti不同，an是 适应在微咸水中生存，在那里美国75%-80%的 制定了蚊子消减计划。

Control of many human diseases is based on control of their mosquito vectors. Biological control programs have been made possible by the recent discovery of Bacillus thuringiensis var. israelensis (Bti) which produces a protein crystal toxic to mosquito larvae. The goals of this project are to: 1) identify which component of the crystal constitutes the actual toxin; 2) characterize the plasmid coding for the toxin; 3) clone the toxin gene into bacteria more suitable for long term mosquito control. Bti has a mammalian and a mosquito toxin within the nine proteins found in purified crystals. No information is yet available regarding the identity of these toxins or indeed whether they are separate toxins. The 9 proteins will be separated by nondenaturing gel electrophoresis and tested for larval toxicity and red blood cell lysis. If the 2 toxins are distinct, it should be possible to obtain mutants or recombinants that produce only the mosquito toxin, thus eliminating potential health hazard incurred by the widespread use of Bti. Both the 4.5 and 72 Md plasmids of Bti have been reported to be the site of the toxin gene. Plasmids will be separated electrophoretically, eluted, and transformed into protoplasts of a plasmid-free Bti. Transformants will be detected by hybridization and tested for toxin gene expression by immunoassay and larval toxicity. If the toxin plasmid is large, it will be digested with restriction enzymes and then ligated until it has been reduced to a more manageable size. The Bti toxin plasmid will then be cloned into 2 more suitable bacteria: 1) Baccillus sphaericus (Bs). Bti does not survive well in nature while the ecological persistence of Bs, also a mosquito pathogen, has been well documented. Accordingly, the Bti toxin gene will be ligated to the pUB 110 plasmid which stably transforms Bs. Hopefully, the recombinant will produce the Bti toxin and maintain the ecological fitness of Bs. 2) The cyanobacterium Anacystis nidulans (An). Bti is less effective against the Anopheles malaria mosquitoes because their larvae are surface feeders while the Bti toxin particles rapidly settle to the bottom. Accordingly, the Bti toxin gene will be ligated to the pUC 104 plasmid which stably transforms An. Gas vacuole-containing strains of An will float at the surface and remain available for Anopheles larvae. Additionally, An, unlike Bti, is adapted for survival in the brackish waters where 75-80% of the U.S. mosquito abatement programs are located.