| This dissertation mainly concerns the construction of genetically engineered Bacillus thuringiensis(Bt) strains with higher toxicity and against a wider spectrum of lepidopteran insects, especially for Spodoptera exigua.The research results are summarized as following:1. The relationship between gene types and the toxicity against S. exiguaIn order to construct satisfactory genetically engineered strain, it is first necessary to understand the relationship between gene types and the toxicity. Nine strains containing crylC were chosen for study for this purpose. PCR amplification was used to detect the ICP(insecticidal crystal protein) genes contained in these strains. According to the amplification results, the nine strains were divided into five types. At the same time, the toxicity of these strains against Spodoptera exigua was tested. The higher potency strains belong to gene types 1 and 2. The strains of gene type 1 contain crylAa, crylAc, cry2, crylC and crylD gene and the strains of type 2 contain crylAa, cry2, crylC and crylD.2. The effects of plasmid pBMBLC (with crylC) on toxicity of natural strains Plasmid pBMBLC was transferred into Bt strain YBT-1535, YBT-1520 and YBT-833.The effects of pBMBLC on these strains were different. The transformants of strain YBT-1535 displayed significantly higher toxicity against S.exigua than strain YBT-1535 (at least 8 times), but the toxicity against Heliothis armigera and Plutella xylostella changed little. For the strain YBT-1520, the effect was not the same as strain YBT-1535. The toxicities of transformants against P. xylostella and H. armigera dropped, and the potency against S. exigua did not change much. Several different transformants were obtained after transferring pBMBLC into strain YBT-833. Transformant YBT-833-1 kept all indigenous plasmids, YBT-833-2 lost a plasmid carrying crylAb and YBT-833-3 lost all indigenous plasmids carrying ICP genes. Comparing their potency with natural strain YBT-833, the overall toxicity of all transformants was not noticeably higher than strain YBT-833. Summarizing the effects of pBMBLC on the three strains, it is known that genetically engineered strain construction using this method can not get satisfactory results, though the potency of YBT-1535's transformants was higher than YBT-1535. Because of the low toxicity level of YBT-1535, the transformants with higher potency cannot be utilized commercially.3. The effect of sporulation-independent promotor on toxicity of natural strainIn order to study the effect of sporulation-independent promotor(P3A), P3A was spliced with the cry1C gene, then inserted into the shuttle vector pHT304, and then recombinated plasmid pBMB827 was obtained. After transferring pBMB827 into strain YBT-1520, it was surprising that the transformants had almost no potency against all lepidopteran larvae tested. And another change was that the spore of the transformant was difficult to release, P3A was not useful in genetically engineered strain construction in this way.Detected by PCR amplification, it was surprising that the transformants of pBMB827 had crylAb specific product, different from their original strain YBT-1520. Result of SDS-PAGE gel electrophoresis proved the existence of crylAb gene. But the reason why pBMB827 could induce expression of the hidden gene crylAb is not clear.4. The development of genetically improved strain YBT-833-2-1 and its specificityBy culturing without antibiotics, one mutant which losing pBMBLC, named YBT-833-2-1, had selected out of YBT-833-2. Activity tests showed that the toxicity of strain YBT-833-2-1 against P. xylostella was higher (P>0.95) than its original strain YBT-833, and its potency against H. armigera and S. exigua increased notably. Compared with the commercial strain YBT-1520, the activity of strain YBT-833-2-1 against H. armigera was lower than the strain YBT-1520, but its potency against P. xylostella and S.exigua was higher than the strain YBT-1520 (P>0.95). According to bioassay results, the overall toxicity of stra...
|
PDF Full Text Request