Cloning, Expression And Insecticidal Activity Of Novel Cry1 Genes From Bacillus Thuringiensis

Bacillus thuringiensis is the most widely applied type of microbial pesticides because its high specificity and environmental safety. The primarily insecticidal protein was Insecticidal Crystal proteins (ICPs)which were encoded by cry or cyt genes. The cry1-type genes were promising tools for effectively controling many species of important lepidopteran pests, but it is difficult to identify known and unknown cry1-type genes rapidly and accurately because of the limitations of identify methods. In order to identify more cry1-type genes, we established a PCR - RFLP identification method of cry1-type genes with high resolution. According to the difference of cry1-type genes'5'and 3'ends, they were divided into four categories and four pairs of universal primers were designed respectively, so the cry1-type genes in the same category could be determined by analysis of RFLP and the resolution is higher. This method could identify cry1-type genes of Bt strains fast rapidly, accurately and effectively, especially identify both known and novel cry1-type genes.By using the original method and new method, three Bt strains which contain many kinds of cry1-type genes were identified respectively. The results showed that the original method could not determine cry1-type genes of Bt strains which contain many genes, and could not determine whether Bt strains contain cry1I genes. The new method could identify three Bt strains which contain many kinds of cry1-type genes accurately, and found some genes that did not find in China, for example, cry1Hb, cry1Ja, cry1Ka, cry1La, and so on. The new method suited for Bt strains which contains one cry1-type gene or many kinds of cry1-type genes.In the identification of the 72 standard strains and 86 Bt isolates, we found 11 standard strains contained cry1-type genes and almost all Bt isolates contained cry1-type genes, and 19 of them were novel. We found other two novel cry1-type genes from Bt standard strains by whole genome sequencing. According to known cry1-type genes'sequences, we designed full-length primers and successfully cloned them except cry1Na, they were designated as cry1Aa16, cry1Ab23, cry1Ab24, cry1Ah3, cry1Ai2, cry1Ba7, cry1Bb2, cry1Be4, cry1Ca12, cry1Da3, cry1Ea9, cry1Fa3, cry1Fa4, cry1Hb2, cry1Ia19, cry1Ib5, cry1Ie2, cry1Ja2, cry1Ka2, cry1La2 by International Nomenclature Committee of Bt. They were successfully expressed in E.coli (Rosetta) by pEB expression vector, expressed products were 60 - 130 kDa. Cry1 protains had insecticidal activity against Plutella xylostella except Cry1Ka2 and Cry1La2. The insecticidal activity of Cry1Hb had not report, but we found it had activity against Plutella xylostella and Heliothis armigera, and the LC50 were 305.82 (241.66 - 391.76)μg/ml and 315.12 (225.68 - 456.86)μg/ml. Cry1Ba7 and Cry1Ea9 had insecticidal activity against Colaphellus bowringi, the LC50 were 5.21 (3.84 - 8.65)μg/ml and 55.78 (43.40 - 88.42)μg/ml, and the activity of Cry1Ea9 against Copeoptera pests was reported firstly. Cry1Ca12 had insecticidal activity against Spodoptera exigua, the LC50 were 89.11 (60.54 - 121.26)×10-3μg/ml. Cry1Ie2 and Cry1Na had insecticidal activity against Ostrinia furnacalis, the LC50 were 0.12 (0.09 - 0.15)μg/ml and 0.15 (0.12 - 0.18)μg/ml.