Construction and expression of prokaryotic expression vector of green fluorescent protein in Brevibacillus brevis and Bacillus thuringiensis isolated from insect larvae intestines

Abstract:

Abstract: [Objective] To construct a prokaryotic protein expression vector of green fluorescent protein (GFP) with the non-sporulation promoter of cry3a gene of Bacillus thuringiensis (Bt), and to obtain recombined engineering bacteria with labled gfp gene and non-sporulation promoter of cry3a gene the construction of insecticidal engineering strains. [Methods] The promoter of gene cry3a and gfp gene was connected by SOE-PCR, The recombined gene pro3a-gfp was inserted into shuttle vector pHT304 at sites BamHⅠand SphⅠ Then plasmid pHT3AG was introduced into Brevibacillus brevis CQUBb and Bacillus thuringiensis CQUBt isolated from Apriona gemari larvae intestines. The expression of the recombinants was analyzed with fluorescent microscope, SDS-PAGE, and then the stability of engineering strains was tested under the conditions with or without antibiotics. [Results] Two engineering strains labeling with GFP were obtained. They expressed green fluorescent protein from the vegetative growth period and about 29kDa green fluorescent protein band was detected by SDS-PAGE. Heterogenous plasmid replicated and expressed stably in engineering strains and did not lead to significant adverse effects on these two host strains. After 30 generations, 95% and 67% of clones was still bearing recombined plasmids respectively. Compared to CQUBt, CQUBb had higher efficiency of electroporation, and the CQUBb-pHT3AG had longer GFP expression time, expressed more protein than CQUBt-pHT3AG, and the stability of CQUBb-pHT3AG was higher than CQUBt-pHT3AG. [Conclusions] We successfully obtained two transgenic insecticidal engineering strains.