Cloning, Expression And Insecticidal Activity Of Cryl Genes In Bacilus Thuringiensis From LaLin Areas In WuChang

Prevention of pest was mainly carried out by means of applying chemical pesticides in the past several decades, which has already throw great wrench into natrual environment and the health of people, as well as led to drug resistance of pest, increase in dozen of drug and cost etc. However, Bacillus thuringiensis as a kind of excellent biological pesticides proves to be safe to environment, human beings and livestock and plays an important role in prevention of pest, and has become the safiest and most widely used microbiological pesticides with the largest production in the world.Bacillus thuringiensis as Gram-Positive bacteria has many advantages, such as specializing in pest very much, harmless to environment. The activity of insect disinfestation is mainly related to gene cry encoding Insecticidal Crystal Proteins, ICPs. Therefore, the study of gene cry has become the focus of recent studies. So far, there has been 605 kinds of insecticidal proteins expressed successfully in the world. But with the further spread of applying Bt, Bt ICPs has caused the resistance of insects to some extent. So seeking for the new strains and genes with higher capacity of toxicity has been become one of the most effective solutions of solving the problems about resistance of insects. Products expressed by gene cry1 is very active in killing various kinds of Lepidoptera pests, and gene crylAc has been studied so deeply that exploration of cryl genes except for crylAc has been paid more and more attentib to, which may provide new alternatives of genes and clash of ideas in such researches.In this study, 3 strains of Bacillus thuringiensis with cryl genes were isolated from soil samples collected in the ares of Lalin town, which is located in Wuchang of Heilongjiang province, they were LB-R-78, LS-R-21, LS-R-30, whose crystals accompanying spore were all rhombic. The 3 strains were identified by means of PCR-RFLP, as a result there were genes such as cry1Ca, cry1Aa, cry1Ab, cry1Ac, cry1Ia, then the whole genes of cry1Ca, cry1Aa, cry1Ab were cloned successfully by using specialized primers of cry1A and cry1C designed and plasmids of LB-R-78, LS-R-21, LS-R-30 as templates, and each gene cloned was subject to the expression experiment in Escherichia Coli. The sequences of the 3 genes above, whose number were HQ412621, HQ685121, HQ685122, respectively, were all registered in GenBank. In addition, the 3 genes were named formally as cry1Ca13、cry1Aa19、cry1Ab25, respectively. The biological activity of Cry1Ca13, Cry1Aa19, Cry1Ab25 protein was tested by turns, and the Plutella. xylostella （Linnaeus） was subject to this test, it was shown that for Cry1Ca13, Cry1Aa19, Cry1Ab25 protein, LC₅₀ was 0.01750μg /ml, 1.18445μg /ml, 1.21566μg /ml,95% confidence intervals was 0.00810～0.02793, 0.97338～1.41618, 0.71699～1.53930, repectively. For positive control Cry1Ac, LC₅₀ was 4.92μg /ml, 95%confidence intervals was 3.80～7.00. All these contributed to the belief that the activity of Cry1Ca13, Cry1Aa19, Cry1Ab25 were all high to Plutella. xylostella （Linnaeus）.