Research On The Relevant Function Gene Of Sporulation And Crystal Formation In Bacillus Thuringiensis

Bacillus thuringiensis(Bt), accounting for 90% in the biological pesticide market, is the most widely used insecticidal micriobe for the high toxin, safety to human beings and animals and no pollution to the environment, etc. Compared with other Bacillus species, producing Insecticidal Crystal Proteins (ICPs) is the notable feature of Bt. B. subtilis, as a kind of mode strain of bacillus, is clearer on the mechanism of spore formation than other Bacillus species, while the study on Insecticidal Crystal Proteins is mainly focused on the expression and regulation of cry gene, the researches on the relation between production of ICPs and genes of sporulation is less. Therefore, two genes located on the chromosome and one of plasmids in Bt were studied by methods of functional genomics in this academic dissertation, and the their effect in the formation of spore and crystal was elaborated, respectively. Experimental results are helpful to clarify the mechanism of the spore and crystal formation, which provides the theoretical basis for constructing the safer and more effective genetic engineering strain.Having made use of Mini-Tn10 carried by the temperature sensitive vector pIC333, we had constructed and screened a B. thuringiensis HD73 transposon insertion mutant library, obtained 20,000 mutants resisted to spectinomycin, and identified mutants completely. Two mutants without ability to produce spores and crystals were isolated. After the analysis of transposon sequence, we found Mini-Tn10 inserted into copG located on chromosome and a gene (named as 11sc) located on a plasmid, respectively, and 9 bp repeated in every insertion site. They were named as HD73(copG)::Mini-Tn10 and HD73(11sc)::Mini-Tn10 respectively.In this study, two knock-out vectors of the ORF of copG partially and completely were constructed, respectively, we named them pRN5101(copGâ–³') and pRN5101(copGâ–³). Then the two vectors were introduced into HD73 strain, which were induced to produce mutants by high temperature. Absent corresponding gene mutants were screened and named as HD73(copGâ–³') and HD73(copGâ–³). The knock-out process of 11sc was the same to copG. Although these mutants produce less ICPs than original strain, they still have the ability to produce spore and crystal.The analysis of the cry1Ac amplification and SDS-PAGE showed that the cry1Ac still existed in these mutants. The phenotype of mutants showed that the deletion of copG and 11sc didn't affect their spore and crystal formation seriously, therefore, they were not the major gene to regulate the spore and crystal formation. In order to explore the effect of the Mini-Tn10 transposon on destruct them and gene expression individually or collectively, further research will be continued on analysis of the genes inserted with the Mini-Tn10 transposon and loss of spore and crystal formation ability.