Study On The Isolation, Identification Of Bacillus Thuringiensis And Cloning, Expression Of Cry Gene

Bacillus thuringiensis (Abbreviation, Bt) as an efficient, safe, pollution-free insect pathogenic microorganisms has been widely used in the biological control of pests. It has been produced largest as a microbial pesticides and widely applied in most area in global. So the study on Bacillus thuringiensis was paid more and more attention by the countries in the world, and the investigation of Bt resources are gradually expanding. Because of the extensive terrain and diversity of environment in our country, it is very significant and necessary to explore the rich Bt resources, screen Bt isolates with high toxicity and clone the novel insecticidal toxin genes for construction of genetically engineered bacteria and transgenic plants. In this study, we described a screening and identification of Bacillus thuringiensis from different sort of soils of Hei Long Jiang Province and Sichuan province, Several novel insecticidal crystal protein genes were cloned and expressed.The concrete results are as follows:1. Three hundred and seventy two soils samples were collected from Hei Long Jiang Province, and 41 strains of native Bacillus thuringiensis were isolated from the samples. The percentage of Bt strains isolation is 11.02%. The most high percentage of Bt isolation from black soil is 15.53% and yellow sand's is lowest as 5.88%, however none Bt strains were isolated from the seventeen saline-alkali soil. So the abundance of Bt distribution have relationship with the Soil kinds. The genotypes of 41 Bt isolates were identified by PCR-RFLP. The results show that 32 isolates harbored cry1 genes(Among them, two strains' restriction map contained a new specific band, speculated that there were unknown genes with them),13 isolates harbored cry2 genes,12 isolates harbored cry1I genes, the known genes were not identified in another nine strains.2. Two hundred and seven soils samples were collected from Sichuan Province, and 26 strains of native Bacillus thuringiensis were isolated from the samples. The percentage of Bt strains isolation is 12.56%. The parasporal crystal shapes of Bacillus thuringiensis were pyramid, cuboidal, round and abnormity, furthermore most of them there were a variety of forms parasporal crystal in one strain.which showed the diversity of Bt resources in Sichuan province.The Identification results of genotype can also display the diversity of Bt strains under the ecological conditions in Sichuan. 26 strains contain cryl, cry2, cry1I and cry9 four genotypes,21 isolates harbored cry1 genes, 8 isolates harbored crylI genes,11 isolates harbored cry2 genes,13 isolates harbored cry9 genes, the known genes were not identified in another five strains. most of them contain a variety of parasporal crystal in one strain. Most of them are crylAa, crylAc, cryllb, cry2Ab and cry9Ba.3. The SDS-PAGE analysis of 67 strains showed that most strains expressed 130kDa protein, and part of them expressed 60,120 and 140kDa protein, a few of them expressed 45,70 and 90kDa protein. Suggesting that they may contain potential novel Cry toxins. There were no proteins expressed in another two strains, the reasons need to be further studied.4. The biological characteristics of Bt strains BF-4 and NJ-1 were studied systematically in this paper. Biochemical reaction indicated that BF-4 was identical with Bt subsp.sotto,while NJ-1 was the same as Bt subsp.galleriae. Crystal shape of BF-4 was bipyramidal, cuboidal and round, but in NJ-1 bipyramidal, spherical and irregular shapes were observed.140kDa ICP was encoded in the two strains, but 60kD ICPs were encoded in BF-4. Three different plasmids were detected from BF-4, there were crylAa,cry1Ac,cryllb,cry2Ab and cry9Ba in the strain. Two different plasmids were detected from NJ-1, there were crylAa, cry1Ia and cry9Ba in the strain..and all genes of these two strains were located on its plasmids seperately.5. Two cry genes cry2Ab and cryllb were coloned from BF-4 successively.They had been registered in GenBank, The accession number is HM037126 and HM051227 respectively, The gene had been named cry2Ab15 and cryllb4 by Btδ-endotoxin gene Nomenclature Committee respectively. Cry2Ab15,70.8kD protein, was composed of 634 amino acids deduced from 1905bps nucletide sequence.Cry1Ib4,81.2kD protein, was composed of 719 amino acids deduced from 2160bps DNA sequences. Isoelectric point of the protein is 6.71, so it is a weak acid protein.Sequence analysis showed that cry2Ab and cryllb are different from the genes published in GenBank.They should be new cry genes.6. cry9Ba2 was coloned from NJ-1.The encoding sequence is made up of 3495 bps, Cry9Ba2,131.6kD protein, was composed of 1164 amino acids. The amino acid sequences deduced from its DNA fragment as 98% homologous with Cry9Ba1. The gene sequence had been registered in GenBank, The accession number is GU299522. The gene had been named cry9Ba2 by Bt 8-endotoxin gene Nomenclature Committee.7. cry2Abl5 and cryllb4 were expressed efficiently in E.coli. Cry2Ab15was highly toxic to the larvae of Plutella xylostella and Helicoverpa armigera, LC50 is 5.7μg/ml and 4.3μg/g respectively. Cry1Ib4 has higer toxic to Plutell axylostella L, but not higher to Colaphellus bowringi L.The Corrected mortality is 82.5% and 46.6% respectively.