| Bacillus thuringiensis (Bt) is the most widely used insecticidal microorganism, which has been successfully used to control the pests in the areas of crop, horticultural crop, forest, and sanitation. Therefore, many countries were interesting to dig the resources of Bt strains and insecticidal crystal proteins. To further dig abundant resource of Bt in our country, screen high toxicity and specific strains, isolate and clone novel pesticidal genes will have important meanings in developing microbial insecticides, constructing high toxicity and broad-spectrum engineering strains and breeding insect resistant transgenic plants. This study described a systematic study of Bt resources in different ecological regions in Sichuan Basin. The concrete results are as follows:1. Sichuan Basin situated in the west of China, the fourth-largest basin of China, is a special area with complicated geomorphology (mountain, pasture, gorge, virgin forest, highland, hurst, glacier, and plain), and contains a rich and unique biodiversity. In total,8418 aerobic sporeformers strains have been isolated from 3834 soil samples collected from different typically ecological regions in Sichuan Basin.13.9% of the isolates were identified as Bt basing on the production of parasporal crystals. A total of 1172 Bt strains have been screened from 3834 soil samples. These Bt strains produced bipyramidal, square, round, and irregular crystal inclusions observed under scanning electron microscopy, indicating the diversity of Bt strains harboured in Sichuan Basin. The analysis of the cry and cyt genes was based on the method of PCR-restriction fragment length polymorphism (PCR-RFLP). cry1, cry2, cry3, cry4/10, cry9, cry8, cry30, cry40, and cyt2-type genes were found in this basin. Strains containing cry1 genes were the most abundant in our collection (66%). Bt strains harboring cry2 genes were the second most abundant (39.4%), and the strains containing cry3, cry4/10, cry9, cry30, and cyt2 genes were found in 1.8,4.1,4.2,3.2, and 1.2%, respectively. The cry8 and cry40 were the least in our collection. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assay of 80 strains which did not produce any PCR products indicated that these strains may harbor potentially novel Cry proteins. Further more, several novel holotype cry genes were found in these strains (JF19-2, BtMc28, Hs18-1, Ywc2-8, and Bm59-2) by the PCR-RFLP and sequencing analysis. In addition, the results of insecticidal activity of some Bt strains showed that strains exhibited a wide range of insecticidal spectrum toxic to dipteran and lepidopteran pests. All these researches mentioned above revealed that the diversity and particularity of gene resources from Bt in Sichuan Basin.2. Cry2-type proteins were different from Cry 1-type not only in structure, but also in pesticidal mechanism. In addition, Cry2 and Cyt protein have the feature of wide range of insecticidal spectrum and enhancing the toxin activity of Cry protein, respectively. First of all, we studied the insecticidal crystal proteins of Bt strains which harbored cry2-type genes. Base on the results of identification of cry 2 and cyt2 gene of all collections in Sichuan Basin, we further analyzed the distribution and gene types of cry2 and cyt2. In addition, two novel pesticidal protein genes were cloned and expressed. The results are as follows:1) 462 Bt strains harbored cry2-type genes, which produced erose, spherical, bipyramidal, and square crystal. The SDS-PAGE analysis of their spore-crystal suspensions revealed that there were four different protein profiles, which had one or two major protein bands with the molecular weights ranged from about 60 to 130 kDa. All these researches mentioned above revealed the diversity and particularity of cry2-type gene resources from Bt strains in Sichuan basin.2) The result of PCR-RFLP showed that Sichuan Basin has three different cry2A-type genes, such as cry2Aa, cry2Ab, a novel cry2A-type gene:the combination of cry2Aa/cry2Ab genes was the most frequent (75.5%), followed by cry2Aa (21.2%) and cry2Ab (3.3%) alone, and one novel type of cry2 gene was cloned from one isolate (JF19-2).3) Only 11 Bt strains contained cyt genes, which was mainly distributed in the soil of forest, and the results of PCR-RFLP indicated that these cyt genes belong to cyt2Aa; 4) The full-length of this novel cry 2A-type gene from JF19-2 and cyt2Aa from BtMc28 was obtained by the method of thermal asymmetric interlaced PCR (Tail-PCR) and PCR, respectively, which were designated as cry2Agl and cyt2Aa3 by the Bt Pesticide Crystal Protein Nomenclature Committee, respectively.5) cry2Ag1 was expressed in E. coli BL21(DE3)pLysS cells, and the results of insecticidal activity assays showed that Cry2Ag1 was toxic to both Dipteran (Aedes aegypti) and Lepidopteran (Plutella xylostella and Helicoverpa armigera) pests. These results strongly suggest that the cloning of the cry2Ag1 gene is not only of interest to researchers studying insecticidal crystal genes, but may ultimately serve as a solution to the increasing resistance of pests to currently used insecticides. However, Cyt2Aa3 was only toxic to Aedes aegypti. These researches are useful for understanding the distribution of cry2-type genes, the features of Bt strains containing cry2-type genes in Sichuan basin, and the insecticidal of the two novel proteins, which may have important meanings in theories and practices.3. Four Bt strains (BtMc28, Bm59-2, Hs18-1 and Ywc2-8) harbored holotype cry genes were isolated from Mu Chuan virgin forest, Bi Feng Gorge natural protection area, and Hai Luo Glacier virgin forest, respectively. In this part, the biological characteristics of these four strains were identified, and the holotype cry genes from these strains were also cloned and successfully expressed in E. coli BL21 (DE3) pLysS cells. All the results are as follows:1) These four strains produced round crystal inclusions observed under scanning electron microscopy; 2) the results of SDS-PAGE analysis revealed that BtMc28 had three major proteins of about 28,70 and 130 kDa, Hsl8-1 and Ywc2-8 both had two major proteins of about 70 and 130 kDa, and Bm59-2 only had about 70 kDa protein.3) The plasmid patterns of these four strains were different from the reference Bt, and the plasmid patterns of Hs 18-1 and Ywc2-8 were similar to each other.4) the results of the identification of flagellum, the physiology and biochemistry test, and the Biolog test of these four strains conformed to the reference Bt strains, but the result of serotype test showed that H antigen of BtMc28, Hs18-1, and Ywc2-8 did not have agglutination reaction with antiserum of reference Bt strains, which indicated that BtMc28, HS18-1, and Ywc2-8 are new H serotypes. In addition, serotype test also showed that Bm59-2 was B. thuringiensis serovar shandongiensis.5) By the methods of SON-PCR, Tail-PCR, and PCR, the full length of 11 novel cry genes were obtained, which were designated as cry56Aa1, cry54Aa, cry54Ba-type, cry53Ab1, cry52Ba1, cry30Fa1, cry30Ea2, cry30Ga1, cry4Cb1, cry4Cb2, and cry4Cc1, respectively.6) cry56Aa1, cty54Aa1, cry52Ba1, cry30Fa1, cry30Ga1, and cry4Cb1 were expressed in E. coli BL21(DE3)pLysS cells. The results of insecticidal activity assays showed that Cry56Aa1, Cry54Aa, Cry52Ba1, Cry30Fa1, and Cry30Ga1 have a wide range of insecticidal spectrum toxic to dipteran and lepidopteran pests. Especially, Cry56Aa1 has the widest insecticidal spectrum, which was toxic to Aedes aegypti, Plutella xylostella, and Helicoverpa armigera. In conclusion, the studying of the biological characteristics of these strains, the analyzing of insecticidal crystal protein genes, and the expression of the holotype cry genes will have important meanings in theories and practices for further using these strains. Our results strongly suggest that the gene is not only a novel resource in the research of insecticidal crystal genes, but it may also serve as an alternative toxin for potential problems associated with insect resistance. The isolation of new Bt strains and toxins continues to benefit the further development of Cry proteins as competitive biological insecticides and breed insect resistant transgenic plants.
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