The Construction Of Marker-free Binary Vector And The Research Of Its Expression System

Usually,we use the resistance-maker-genes to screen the transformants.However,it has become the issue that limit the application of genetically modified Lilium and other plants widely,as it is responsible with the safty of the genetic modified organisms to some extent.Besides a few ofresistance-maker-gnes can make goal-genes silence.Recently we have two approaches to solve the problem.One is no resistance-maker-genes,the safe-maker-genes or marker-free-genes can be used as screening the transformants;the other one is no marker genes.we firstly screen the transformants using resistance marker genes and then have these maker genes kicked out.We constructed two marker-free binary vectors using these two different approaches,and inoculated tobacco with one of the vectors via Agrobacterium infection and transformation.This research can not only advance the development of gene transformation,but also can make gene safety issues less difficult.The 823 bp upstream regulatory region of RD29A gene from Arabidopsis genome was isolated by PCR technique.Sequence analysis showed that the cloned fragment shared 99%identity with the reported RD29A promoter which contained several cis-acting elements involved dehydration-responsive elements(DRE)and ABA responsive elements(ABRE).The gene expression responds to drought,low temperature and ABA treatment.One of the vectors is the the binary vector in which IPT gene expression was driven by RD29A promoter.The IPT(isopentenyltransferase) gene was isolated by PCR from related plasmid. IPT gene has been used as safe selection marker gene in plant genetic engineering.In this paper the binary vector in which IPT gene expression was driven by RD29A promoter was constructed.We also constructed the vector PG-35S-IPT.as a contrast.Another one is screening the positive transformants by virtue of excising the selectable marker genes.We studied the site-specific recombination technic of Cre/Lox system.We use promoter RD29A drive the expression of the gene Cre.Firstly,we put the Pnos:NPTâ…¡: nos between two Lox sites which are in the same direction,then constructed several middle vectors,and finally completed the construction of Pksb-RD29A-Cre/Lox system.So we can use the selectable marker gene to screen the positive transformants in the first stage and then excise the selectable marker gene via low temperature inducing or ABA or other ways to acquire the positive transformants without the selectable gene.This working model mediated by Cre/Lox system should be useful for the improvement of the present plant transgenic technology.In the last part of this project,we did experienments using tobacco which was inoculated with this binary-vector PG-RD29A-IPT and PG-35S-IPT via Agrobacterium infection and transformation.After that,we studied the different treatment conditions that concluded the effect of pre-culture time on gene transformation and effect of infection time of Agrobacteria and the low temperature on RD29A and 35S. And wediscussed the possibility of marker free gene system PG-RD29A-IPT.Finally,we identified the positive transformants of tobacco by PCR.