Research On Labeling Resident Bacteria Isolated From Insect Larvae Intestines With Green Fluorescent Protein And Its Expression Characteristics

Bacillus is a very potential host bacterial, which can be used to study protein expression system, construction of biocontrol bacteria and so on. In this study, we constructed a prokaryotic protein expression vector of green fluorescent protein (GFP) with the non-sporulation promoter of cry3a gene of Bacillus thuringiensis (Bt), obtain recombined engineering bacteria with labled gfp gene and non-sporulation promoter of cry3a gene which will lay the foundation for localize the bacteria in insect intestines and the construction of insecticidal engineering strains. The promoter of gene cry3a and gfp gene were connected by SOE-PCR, The recombined gene pro3a-gfp was inserted into shuttle vector pHT304 at sites BamHⅠand SphⅠ. And then plasmid pHT3AG was introduced into Brevibacillus brevis CQUBb and Bacillus thuringiensis CQUBt, which were isolated from Apriona gemari larvae intestines, respectively by electric translation. The expressions of the recombinants were analyzed with fluorescent microscope, SDS-PAGE, and then the stability of engineering strains was tested under the conditions of antibiotics and without antibiotics. These are the major results of this study:Two engineering bacteria strains labeling with GFP were obtained. They can constitutive express green fluorescent protein from the vegetative growth period and about 29 kDa green fluorescent protein band was detected by SDS-PAGE. Heterogenous plasmid can replicate and express 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. Compare to CQUBt, CQUBb had higher efficiency of electroporation, and the CQUBb-pHT3AG has longer GFP expression time, much more express protein than CQUBt-pHT3AG, and the stability of CQUBb-pHT3AG was better than CQUBt-pHT3AG. We successfully made the promoter of cry3a work in Bt strains (CQUBt) and non-Bt Strains(CQUBb)and obtained two transgenic bacteria strains which can express GFP efficiently and can be used in locating, intestinal microecology and the construction of Bacillus expression system, insecticidal engineering strains.To identify the heredity stability of a recombinant soluble expression vector pHT3AG in wild Brevibacillus brevis and to test he stability of engineering strain after freezing storage. The heredity stability of pHT3AG was analyzed by continual subculture. And the determination of exogenous plasmid copy number and stability using were conducted using relative quantitative real-time PCR. Quantitative real-time PCR which was a accurate, rapid, convenient, high-throughput technology is chosen for this purpose of plasmid copy number determination. It was segregationally unstable of the plasmid under no erythromycin selection pressure, only 40% cells keeping stable after 100 generations. But it could be controlled by the supplementation of selection pressure, kept 55% after 100 generations. The growth characteristics and the morphology of the cultures and the sequences did not show differences among the generations. GFP expression level showed no difference among the generations by SDS-PAGE.In the determination of PCN using qPCR, to avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated with Ultrasonic cell disruption Miriam for 10 minutes and then treated by heating at 98℃prior to storage at -20°C, were used as the template of qPCR. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. The method was applied to study the PCN of recombinant Brevibacillus brevis. After this determination the PCN of our strains in subculture processes, the PCN had a downward trend which can demonstrate the stability of plasmid. Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination of recombinant Brevibacillus brevis in fermentation processes and subculture processes. Under the selective pressure of antibiotics, the plasmid of our subcultured strains was more stable than strains without selective pressure of antibiotics.Final concentration of glycerol 20% or 30% and preservation temperature -40℃was the best condition on which the stability was more than 50% after 12 month freezing storage. The growth characteristics and protein expression wasn't different from strains recovered successfully. It is indicated that recombinant strain has higher heredity in 40 generations or fermented with selection pressure. The Brevibacillus brevis strain can be used in construction of protein expression system and so on.