Primary Studies On Construction Of The Chloroplast-nuclear Co-transformation Vector And Transformation On Nicotiana Tabacum L.

Up to the present, technique for transgenic plants have developeda lot, there are many mature and effective methods to select andregenerate transgenic plants. Now the selectable marker genes (SMGs)for transgenic plants always base on antibiotic resistance genes. SMGsare used for negative selection and proved to be efficiency for enablingplant transformation, however, SMG-transgenic plants have potentialrisks to human body and the environment, This is also one of the reasonswhich prevents the application of transgenic plants in practice.Moreover, presence of the SMGs can prevent repetitious transformationusing the same marker gene. Removal of the SMGs also could reduce targetgene silence. So researches about the control of the SMGs especiallythe antibiotic resistance genes have developed rapidly. Until todaythere are many strategies for the security of transgenic plants. Oneof the methods is to delete the SMGs through co-transformation afterobtaining the transgenic plants. Until now these methods are based onco-transformations of target gene and marker gene to the nuclear genomeof plants. Report about the co-transformation of chloroplast genome and nuclear genome has not been announced.Our research is based on establishing a model for biologic securityof the transgenic plants. This article will discuss the possibility ofobtaining the marker-free transgenic plants through the mode ofco-transformation of chloroplast-nuclear genome by constructing thechloroplast-nuclear transgenic vector and transforming the model planttobacco.First, a mgfp4 expression cassette CaMV 35S Pro-mgfp4-NOS ter wascloned into the plasmid pICF6061 to construct a preliminarychloroplast-nuclear genomic co-transformational vector pICF6061. G.Then a matrix association region(MAR) was amplified by PCR from tobaccototal DNA. It was confirm a tobacco MAR sequence Rb7by sequence analysisand blast in NCBI and DNAman. Rb7 was cis-inserted into the vectorpICF6061.G by flanking the mgfp4 expression cassette and constructeda ultimate chloroplast-nuclear genomic co-transformational vectorpICF6061.G.R. The pICF6061. G.R included a chloroplast genomicexpressing gene aphA-6as a kanamycin resistant selectable marker. TheaphA-6 expression cassette possessed a tobacco chloroplast genomic 16SrRNA promoter. The pICF6061. G.R also included a nuclear genomicexpressing gene mgfp4, which possessed a tobacco nuclear genomic 35Spromoter. Flanking the aphA-6and mgfp4 cassettes there are two tobaccochloroplast sequences trnN and trnR, which could enable the foreigngenes to the tobacco chloroplast genome by homologous recombination.MAR sequence Rb7 which flanking the foreign gene can minish the foreigngene expressing differentia between different transgenic plants. So itcould promote the expressing level.of mgfp4.Bombardments were carried to the three-month-old tobacco leavesusing the gold microcarriers coated with chloroplast-nuclear genomicco-transformational vector pICF6061. G.R through biolistic gun. Thebombarded leaves were cut into small pieces on 400mg/L kanamycin-containing regeneration medium. These small pieces of leavesregenerated to forming callus, shoot and cluster shoot orderly.Regenerated shoots were rooted on phytohormone-free MS medium in thepresence of kanamycin. RT-PCR indicated that aphA-6was recombined intochloroplast genome and gave the expression. Regenerated kanamycinresistant tobacco was checked for the expression of mgfp4 byfluorescence microscope and chimerism of green fluorescence expressionwas observed in the leaf cell. RT-PCN indicated that mgfp4 was expressedin tobacco nuclear.Our research showed that using one vector for thechloroplast-nuclear genomic co-transformation through biolistic gunwas conceivable. Farther researches should be done to discuss theefficiency of obtaining marker-free transgenic plants by onetransformation and succedent hybridization with female wild type basedon this conclusion. Nevertheless possibility of transfer events fromchloroplast genome to nuclear genome was considered yet.