Homology Analysis And Construction Of Gene Library Of 20kb DNA In Parasporal Crystal From Bacillus Thuringiensis Strain 4.0718

Bacillus thuringiensis has been widely used for insect control because of the specific larvicidal activity of parasporal crystals produced during sporulation.It was proved that the parasporal crystals contained 20kb DNA fragments intimately associated with protoxins,which play a important role in the assembling and stability of parasporal crystals as well as the activation of crystal toxin in the midgut of target larvae.However,the whole sequence of 20kb DNA was unclear to date.The present investigation constructed 20kb DNA gene library to discover more sequence information and further determined the origin of DNA.The data will be helpful for further understanding the mechanism of interaction between Bt protoxin and DNA as well as the role of the DNA in the structure and fuction of Bt crystal protein.In this research,we took Bt strain 4.0718 as material and isolated 20kb DNA from its parasporal crystals.We constructed gene library using DNA fragments of Sau3AI partial digestion and pbluescriptâ…¡(SK+),vector.The number of recombinants which were screened out was 245,yielding a genomic coverage of five and concluding 99%sequence of DNA which achieved the requested theoretical value to construct gene library.Then, we checked randomicity,average length of insert fragments and empty vector rate of gene library.By NCBI Blast,the.sequencing results proved that the DNA came from genome of Bt.Moreover, we found a series of genes located in 20kb DNA for the frist time.This research laid a basis for future understanding the mechanism.of interaction between Bt protoxin and DNA as well as the the origin,structure and fuction of DNA.