The Construct Of Bacillus Amyloliquefaciens Barnase-barstar Molecular Regulation System And Application Research In Tobacco Flower Development Regulation

Barnase is a cell lethal factor derived from Bacillus amyloliquefaciens, this geneexpression in the inflorescence meristem and stated by the inflorescence meristempromoter can kill the cells so inhibiting plants flowing, then the plant can not enterthe reproductive growth and long-term in nutritional growth status. Barstar is theinhibitory factor of Barnase, the expression of barstar can prevent the lethal effect ofbarnase, then the plants can recover their fertility and enter into reproductive growthstates produce seeds. Barstar started by a chimeric promoter MRE35S90, expressionof this gene is controlled by applying chemical agent copper ions, when manuallyadding the inducer, the inducer will be binding with ACE1, then will let the responsepromoter express barstar.The plant reproductive organs constructed in this test can express in a particulartime and space, this system can be controlled according to the needs of people.We obtained barnase and its suppressor gene barstar through culturing Bacillusamyloliquefaciens, extracting genomic, designing primers and PCR; obtaininflorescence meristem-specific promoter AtREM1by culturing wild-typeArabidopsis thaliana, extracting total genomic designing primers and PCR, thispromoter can start barnase expression; obtain rbcs3A terminator from wild pea(ViciaLinn) ACE1and NOS terminator from yeast (Saccharomyces cerevisiae) by samemethods; synthetic MRE35S90and CaMV35S promoter start barstar and ACE1expression.Combined each gene in an expression plasmid named RBCu, transform thisrecombinant plasmid into Agrobacterium tumefaciens LBA4404, then transformhigh-quality tobacco varieties NC89to obtain the transgenic tobacco.Experimenter use herbicide-resistant screening, PCR, Southern blot, andreal-time quantitative PCR and other technical means to detect the transgenic tobaccoand copy number of the target gene, those methods can detect the transgenic tobaccoon integration level, transcription level and expression level.Paper tests completed floral retarded system and chemical-induced floral retarded recovery system construction, the system can be applied to other plants toprotect the environment and enhance the economic crops or crop yield, promote theeconomic and human society development to a certain extent.