Characterization of insecticidal crystal proteincry gene ofBacillus thuringiensis from soil of Sichuan Basin, China and cloning of novel haplotypescry gene

Abstract

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 order to describe a systematic study of cry gene resources from Bacillus thuringiensis (Bt) strains of different ecological regions in Sichuan Basin, a total of 791 Bt strains have been screened from 2650 soil samples. The analysis of the cry genes was based on the method of PCR-restriction fragment length polymorphism (PCR-RFLP). cry1, cry2, cry3, cry4/10, cry9, cry30, and cry40-type genes were found in this basin. Strains containing cry1 genes were the most abundant in our collection (66%), and twenty-one different cry1-type gene combinations were found. Bt strains harboring cry2 genes were the second most abundant (39.5%), and the strains containing cry3, cry9, cry4/10, cry30, and cry40 genes were found in 2.5, 3.5, 4.2, 4.2, and < 1%, respectively. Furthermore, several novel haplotypes cry genes were found, and the full-length sequences of three novel cry genes were obtained, which were designated as cry52Ba1, cry54Aa1, cry30Fa1 by the B. thuringiensis Pesticide Crystal Protein Nomenclature Committee, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assay of 80 strains which did not produce any PCR products indicated that these strains may harbour potentially novel Cry proteins. All these researches mentioned above revealed that the diversity and particularity of cry gene resources from B. thuringiensis strains in Sichuan Basin.

Characterization of cry2-type genes of Bacillus thuringiensis strains from soil-isolated of Sichuan basin, China

“…These distinctive features and diversity of insects provide the opportunity of isolating novel entomopathogenic bacteria, so it is most possible that some novel cry2 genes or special Bt strains may be found. Our lab had surveyed the distribution of cry gene in Sichuan basin (25). In the present study, we specially identify the distribution of cry2 genes and the type of cry2 genes in this basin.…”

Characterization and expression of cry4Cb1 and cry30Ga1 from Bacillus thuringiensis strain HS18-1

“…The SEM and SDS-PAGE analysis of HS18-1 were performed as described by Zhu et al (2009) and Ibarra et al (2003), respectively.…”

Characterisation and expression of a novel holotype crystal protein gene, cry56Aa1, from Bacillus thuringiensis strain Ywc2-8

“…The sample was then sputter-coated with gold in an IB-5 ion coater (HITACHI) for 5 min. The SEM micrograph was taken as described by Zhu et al (2009).…”

“…As such, it contains the richest and widest biodiversity. These distinctive features and diversity of insects provide the opportunity to isolate novel Bt strains and cry genes (Zhu et al 2009). Recently, we isolated a novel Bt strain (Ywc2-8), native to the Sichuan Basin, which exhibited a wide range of insecticidal activity (i.e., it was toxic to Dipteran and Lepidopteran pests, data not shown).…”