Construction And Function Analysis Of Heat-induced Selectable Marker Gene Excision System

Since scientists firstly got transgenic plant in 1983,the transgenic technology has been widely used in the study of changing the inherited traits of plant.In plant transformation systems,selectable marker genes,which confer resistance to selective chemical agents such as antibiotics or herbicides, are often required to select transformants from non-transformed plant cells and tissues during the process of production of transgenic plants.For example the plants with nptⅡgene has resistence to kanamycin.However selectable marker genes conferring antibiotic or herbicide resistance,used to introduce economically valuable genes into crop plants,have four major problems:(1) the selective agents have negative effects on proliferation and differentiation of plant cells,(2) there is uncertainty regarding the environmental impact of many selectable marker genes,(3)there are concerns in terms of food safety and consumption of transgenic food as well.(4) it is difficult to perform recurrent transformations using the same selectable marker to pyramid desirable genes.In fact,the selectable maker gene which is no longer necessary to transgenic plant after selecting transgenic cells should be removed.So far several approaches to generate marker-free transgenic plants have been reported, such as transposon system,homologous recombination,co-transformation,site-specific recombination systems of prokaryotic or yeast(R/RS,Cre/LoxP,FLP/FRT) et al.Among these approaches,the site-specific recombination system is universal used,but there are also some disadvantage in this system(such as lack of stabilization,difficulty in excision or incomplete excision),therefor there is great necessity to make it perfect.In this study,we utilized tight heat-stock promoter HSP70m to control Cre/LoxP recombination system,simultaneity because the constitutive expression of ipt could cause the generation of a lot of abnormal adventitious shoots,we utilized this trait to generate adventitious shoots in the medium without exterior hormone,and used ipt gene and nptⅡgene as selectable genes to make sure that the plants we got were transgenic.After we got transgenic plants,through heat-stock treatments,the ipt,cre and nptⅡgenes were removed,and the adventitious abnormal shoots caused by the constitutive expression of ipt generated phenotypically normal shoots gradually,and we got maker-free transgenic plants.The main results are as follows:1.Cloning of ipt and creDesigned two pairs of specific primers P3/P2 and P4/P5,and then utilized pfu DNA polyase to amplify the ipt fragment and cre fragment from Agrobacterium tumefaciens C58 and Escherichia coli BM25.8 genome separately.The results showed that the sequences of genes cloned were equal to the reported in GenBank and demonstrated that the genes we cloned were ipt and cre.2.Construction of heat-induced selectable marker gene auto-excision system with ipt and nptⅡas selectable marker genesWe successfully constructed three vectors pQSL05,pOSL15 and pQSL16,pQSL05,in which there is a 35S-ipt fragment but not nptⅡgene,was used to measure the function in higher plants of the cloned ipt and the selectable efficiency of ipt as single selectable maeker gene;the vector pQSL15 was utilized to confirm the efficiency of excision of the selectable maker gene nptⅡof the site-specific recombination system Cre/LoxP under the condition that cre was controled by the heat-stock promoter HSP70m.And the last vector pQSL16 was also called cre/LoxP-ipt-MAT(cre/LoxP-ipt type MAT),which contains nptⅡ,ipt with native promotor and terminator,and Cre/LoxP site-specfic recombination which controled by heat-stock promotor HSP70m,was used to confirm the effect of ipt,double-selectable system and the heat-induced selectable marker gene auto-excision system in transgenic tobacco.3.Heat-induced selectable marker gene auto-excision system had a positive function in transgenic tobaccoTobacco leaf disc was infected by Agrobacterium tumefaciens EHA105 harboring pQSL05, pQSL15 and pQSL16 separately,and obtained three kinds of transgenic tobacco.(1) the transgenic plants which infected by Agrobacterium turnefaciens EHA105 harboring pQSL05,because of the constitutive expression of ipt,lost apical dominance and rooting ability and generated a lot of abnormal adventitious shoots without the application of exogenous cytokinins.Extracted total DNA of the abnormal adventitious shoots,and obtained ipt fragment through PCR.All the evidences demonstrated that ipt gene was introduced into tobacco genome and was successfully expressed and showed that the ipt could not be used as selectable gene singly.(2) the transgenic plants which infected by Agrobacterium turnefaciens EHA105 harboring pOSL15,which had green fluorescence when they were cultured in MS medium with 150mg/liter kanamycin at 25℃,and it was showed that the T-DNA of pQSL15 was integrated into tobacco genome.After heat-stock treatment,the adventitious shoots cultured in MS with kanamycin died gradually,but the ones cultured in MS without kanamycin did not die but they did not had green fluorescence no longer.This result show that nptⅡand gfp were removed from the genome by heat-stock treatment.Then we extracted the total DNA of the transgenic plants cultured in MS medium with kanamycin at 25℃and in MS without kanamycin under a temperature-period of 30℃15 h/35℃9 h,and we could only get nptⅡand gfp from the plant cultured at 25℃through PCR.The evidences of phenotype and molecule showed that the selectable maker gene nptⅡand gfp was excised mediated by cre/LoxP site-specific recombination after heat-stock treatment.(3) the transgenic plants which infected by Agrobacterium turnefaciens EHA105 harboring pQSL16,generated the similar abnormal adventitious shoots with the transgenic plants which infected by Agrobacterium tumefaciens EHA105 harboring pQSL05 in MS medium with kanamycin but without hormone.After heat-stock treatment,the adventitious shoots cultured in MS medium with kanamycin gradually died,the ones in MS medium without kanamycin showed differernt phenotype:the new birth shoots were normal,and they also could generat roots and the roots didn't have green fluorescence.Then we isolated total DNA of the transgenic plants cultured in MS medium with kanamycin at 25℃and in MS without kanamycin under a temperature-period of 30℃15 h/35℃9 h,and we could only get nptⅡand gfp from the plant cultured at 25℃through PCR.All the results confirmed that the heat-stock-cre/LoxP-ipt-MAT vector system provides a promising way to obtain free-maker transgenic plant in the medium without hormone.