Aims: To isolate and characterize the novel Bacillus thuringiensis strains from bryophytes collected from Wuyi Mountain, Fujian Province of China, and identify new B. thuringiensis strains and toxins active against mosquitoes. Methods and Results: Twelve novel B. thuringiensis strains were isolated from 76 bryophyte samples. According to the results of this preliminary screening, LLB6 was the most toxic to Aedes albopictus. Then phase-contrast as well as scanning electron microscopy, bioassays, cloning, sequencing and expression were performed to characterize the novel isolate LLB6 and its new gene cry2Ac5. Conclusions: Bacillus thuringiensis occurred naturally on bryophytes. LLB6 isolated from Physcomitrium japonicum was toxic to A. albopictus. A new cry2Ac5 gene of LLB6 was detected, cloned and expressed successfully. Bioassays on A. albopictus showed that the expressed Cry2Ac5 was also toxic to the third instar larvae. Significance and Impact of the Study: This is the first report of B. thuringiensis strains isolated from bryophytes. It represents a specific source of new B. thuringiensis strains and is of great importance for the knowledge of the ecology of B. thuringiensis. Novel LLB6 harboring the new gene cry2Ac5 and its expressed Cry2Ac5 protein revealed activity against A. albopictus and became a new member of B. thuringiensis toxins. 301Japanese encephalitis all over the world. About 20 million people are infected by dengue virus transmitted by Aedes mosquitoes with about 24 000 deaths every year. Therefore, mosquito management is an essential component for the control of mosquito-borne diseases. It has been known that the cyt and cry genes (cyt1, cyt2, cry2, cry4, cry10 and cry11) of B. thuringiensis encode the proteins active against mosquitoes. However, insect resistance to B. thuringiensis, especially to Cry proteins, has been found in recent years. Moreover, the similar or even higher level of insect resistance to commercial B. thuringiensis products has been reported in many countries. As a result of the severe impact of mosquito-borne diseases on public health, identification of new B. thuringiensis strains and toxins active against mosquitoes has increasingly come to attention (Schnepf et al. 1998).Wuyi Mountain is one of the world's natural and cultural heritages in China. It has a typical moderate subtropical climate with abundant rainfall, and warm and humid weather. The bryophyte samples collected from Wuyi Mountain were used to isolate B. thuringiensis. The purpose was to gain more information on the distribution of B. thuringiensis populations and to screen the strains active against A. albopictus. LLB6 was obtained and found to contain the cry2Ac gene, encoding protein toxic to Dipteran insects. To learn more about the characteristics of the cry2Ac gene of LLB6 and its encoding protein, we cloned and sequenced this gene, and successfully expressed the protein in Escherichia coli BL21. The Cry2Ac of LLB6 had been named as Cry2Ac5 by the B. thuringiensis delta-endotoxin nomenclatu...
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