Effect Of The Sigma35 Factor On The Expression Of The Cry Gene In Bacillus Thuringiensis

Bacillus thuringiensis is a soil bacteria which can produce spores while generate a lot of insecticidal crystal proteins in forms of parasporal crystal in the stationary phase. The mechanism of highly efficient protein expression and insecticidal had attracted a lot of researchers'interesting. However, the molecular mechanism of formation of crystal proteins is not clear. In order to figure out the effect of Bacillus thuringiensis spore-specific transcription factor-Sig35 on the expression of spores and insecticidal crystal protein, we carried out some work which based on Bacillus thuringiensis (subsp.kurstaki) HD-1 in this text.1. Construction of Bacillus thuringiensis shuttle vector pHT1K-Plip-TS. We cloned the non-spore-specific strong promoter of Bacillus subtilis and the terminator gene of cry1Ac into the shuttle vector pHTIK of Bacillus thuringiensis to construct the shuttle vector pHT1K-Plip-TS. The promoter HpaⅡcould activite expression of genes from the vegetative phase of Bacillus thuringensis. The structure of cry1Ac terminator can make mRNA more stable. So we can make genes expresse in the early stage in Bacillus thuringiensis and accumulate a large number of proteins as a consequence.2. Construction ofσ35 gene overexpression recombinant strain. First, we cloned the sig35 gene into shuttle vector pHT1K-Plip-TS to constructσ35 gene overexpression vector pHT-1K-Plip35TS. Then the vector was transformed into Bacillus thuringensis subsp. kurstaki (HD-1), and named 35/kur. So 35/kur strain could expresseσ35 independent on spore to from theσ35 factor at vegetative phase of Bacillus thuringensis.3. Detective of the translation of cry gene. Compared with the wild strain kurstaki (HD-1), expression level of the crystal protein in theσ35 overexpression strain (35/kur) is higher. The results of RT-PCR show that transcription ofσ35 in over-expression strain initiate at vegetative phase, while the wild strain at the stationary phase. And the transcription of cry1Aa advanced to vegetative phase in the recombinant strains. However, expression of Cry1Aa protein was not detected in the early stage on the translation level until the stationary phase. We speculated that experssion of crystal protein need the regulation of other genes as complemention. Increase ofσ35 factor in the vegetative phase could relieve the competition of the genes which have common promoter region but the regulation at post-transcriptional level has not been activated at the same time.