Researching On The Plasmids Of Bacillus Thuringiensis HD73Strain And Its Deletion Mutants

Bacillus thuringiensis is an important insecticidal microbial because of itsspecific-insecticidal and high-efficient acvitiy. Its insecticidal genes usually habor thelarge plasmids, Thus, sequencing and researching the plasmid is help us betterunderstand the relationship between the plasmids and the insecticidal genes and therole of plasmids in bacterial growth and development.By the454and Solexa high-throughput sequencing technology, the sequencesof two large plasmids pHT73plasmid (carrying cry1Ac gene) and pAW64plasmidwere assembled. Sequence analysis showed that the pHT73of plasmid of77437bpsencoded77ORFs.The RT-PCR analysis, indicated that61ORFs of63ORFs detectedshowed transcriptional activity at the T-1, T0, T7(T0is symble for the end ofexpotential phase);the pAW64plasmid of76490bps, encoded93ORFs,and RT-PCRanalysis demonstrated that91ORFs T-1, T0and T7, had transcriptional activity. Inaddition, the sequence assembly of the four small plasmids in the HD73strain wasperformed, sequence analysis showed that p1plasmid of11769bps, p2plasmid of8513bps, p3plasmid of7635bps and p4plasmid of8241bps encoded26ORFs,16ORFs,10ORFs, and10ORFs, respectively.In order to study the impact of the plasmid on the growth and development ofHD73strain, four mutants with various plasmid deletions were constructed by usingof high-temperature curing plasmid method and verified by PCR, and two mutantswith plasmid deletions were identified from the stored strains in the laboratory. Nosignificant differences in the growth rate, spore germination, and spore productionwere found among wild-type strain HD73and six mutants. However, the lack ofplasmids effected on bacterial color and biomass settling velocity. The results showedthat the bacteria with the absence of p4, pHT73plasmid grew yellow on LB agar plateand the absence of p3、pAW63、pHT73plasmid promoted bacterial biomass settlingvelocity. In order to study the role of pHT73plasmid on the cry1Ac gene expression, acry1Ac gene deletion mutant HD△cry1Ac was constructed through the homologousrecombination method.The results showed that the deletion of the cry1Ac gene had noeffect on the growth conditions, spore germination and spore production.The vectorpHT315-cry1Ac-gfp with cry1Ac and gfp fusion was transferred into HD73-T4andHD△cry1Ac strains, respectively. The observation by the laser scanning confocalmicroscope showed that the cry1Ac gene expression had no significant difference inthe HD73-T4、HD△cry1Ac strains.The sequence assembly of the pHT73, pAW64, p1, p2, p3, p4plasmid, wasfinished in this study. It will provide a basis for further study on the function and roleof these plasmids.