Kucińska Jolanta, Lonc Elzbieta, Rydzanicz Katarzyna
Zakład Parazytologii Ogólnej, Instytut Mikrobiologii, Uniwersytet Wrocławski.
Wiad Parazytol. 2003;49(1):11-20.
Identification of Bacillus thuringiensis (Bt) parasporal crystalline inclusions composed of Cry proteins (=delta-endotoxins) resulted in introduction of microbial pesticides for biological control of some parasites. Delta-endotoxins are encoded by cry genes and are active against pest and nuisance insects (mostly mosquitoes and black flies--vectors of still important infectious diseases). The recent significant progress in DNA recombination technique may overcome limitations (a short residual persistence and a narrow spectrum of activity) associated with application of Bt conventional products. An introduction of cry genes from mosquitocidal subspecies B. th. israelensis (Bti) to the aquatic microorganisms inhabiting the same water bodies as mosquito and fly larvae (Diptera), has considerably improved the toxin delivery system to target insects. However, in the first experiments, in which Bti genes were cloned in cyanobacteria (Agmenellum quadruplicatum, Synechocystis PCC6803), a low gene expression was observed. Thus, it was necessary to integrate cry genes with strong promoters or to increase the number of vector-introduced copies. To overcome the obstacles of low gene expression and regulatory restriction for recombinant organisms, Bti spore/crystal formulations were encapsulated in the aquatic protozoan, Tetrahymena pyriformis. Large numbers of crystals (180 to 240/cell) were accumulated in its food vacuoles. This system resulted also in an increase in toxin persistence from 24 to 71 h. Cloning Bti genes in B. sphaericus (which also produces mosquitocidal proteins) was another way of an increasing Bt crystal residual activity. In this case, the crystals were additionally protected by B. sphaericus exosporium. These transgenic bacteria produced large amounts of delta-endotoxins that remained under water surface longer than the wild B. sphaericus strains. Moreover, they had a broader spectrum of insecticidal activity, because B. sphaericus is toxic mostly to Culex and Anopheles, and Bti--mostly to Culex, Aedes and some Simmulidae. Gram-negative bacteria (Asticcacaulis excentricus, Caulobacter crescentus and Ancylobacter aquaticus) turned out also to be effective delta-endotoxin producers. They grow on simple media and do not contain proteases which could degrade Cry proteins. In some cases, 100% mosquito larvae mortality was observed as a result of an exposure to transgenic microorganisms containing Bti genes. However, transgenic techniques are still not very popular in the world, despite their efficacy in biological control of insects. The transgenic organism construction is expensive and time-consuming. Genetic engineering is still raising a lot of anxieties and doubts concerning inappropriate use of modified organisms. On the other hand, this technology could solve many problems associated with vectors of important diseases, which are still unapproachable to contemporary medicine.
苏云金芽孢杆菌（Bt）伴孢晶体包含Cry蛋白（即δ-内毒素），这一发现使得微生物农药得以引入，用于生物防治某些害虫。δ-内毒素由cry基因编码，对害虫和滋扰性昆虫（主要是蚊子和蚋——仍然是重要传染病的传播媒介）具有活性。DNA重组技术最近取得的重大进展可能会克服与Bt传统产品应用相关的局限性（残留持久性短和活性谱窄）。将杀蚊亚种以色列亚种Bt（Bti）的cry基因引入与蚊子和苍蝇幼虫（双翅目）栖息在同一水体中的水生微生物，极大地改善了毒素向目标昆虫的递送系统。然而，在最初的实验中，当Bti基因克隆到蓝细菌（四倍体阿格门氏菌、聚球藻PCC6803）中时，观察到基因表达水平较低。因此，有必要将cry基因与强启动子整合，或者增加载体导入的拷贝数。为了克服重组生物基因表达低和监管限制的障碍，Bti孢子/晶体制剂被包裹在水生原生动物梨形四膜虫中。大量晶体（180至240个/细胞）在其食物泡中积累。该系统还使毒素的持久性从24小时增加到71小时。将Bti基因克隆到球形芽孢杆菌（它也产生杀蚊蛋白）中是提高Bt晶体残留活性的另一种方法。在这种情况下，晶体受到球形芽孢杆菌外孢子的额外保护。这些转基因细菌产生大量的δ-内毒素，在水面下停留的时间比野生球形芽孢杆菌菌株更长。此外，它们具有更广泛的杀虫活性谱，因为球形芽孢杆菌主要对库蚊和按蚊有毒，而Bti主要对库蚊、伊蚊和一些蚋科昆虫有毒。革兰氏阴性细菌（偏心柄杆菌、新月柄杆菌和水生柄杆菌）也被证明是有效的δ-内毒素生产者。它们在简单培养基上生长，并且不含有可降解Cry蛋白的蛋白酶。在某些情况下，由于接触含有Bti基因的转基因微生物，观察到蚊子幼虫死亡率达到100%。然而，尽管转基因技术在昆虫生物防治方面有效，但在世界范围内仍然不太受欢迎。转基因生物的构建成本高昂且耗时。基因工程仍然引发了许多关于不当使用改良生物的焦虑和疑虑。另一方面，这项技术可以解决许多与重要疾病传播媒介相关的问题，而这些问题是当代医学仍然无法解决的。
