Construction Of Tobacco Chloroplast Transformation Vector With The Novel Selecting Marker

Several genes encoding the enzymes that decompose the antibiotics are applied as the selecting markers of chloroplast transformation.They display efficient in the screen of transience plants,However,these genes have sevsral disadvantages.Non-antibiotic genes have been applied in recent years.Among them,some genes encode the enzymes involved in the biosynthesis of plant tetrapyrroles including hemes,chlorophylls and siroheme, which are important for plant growth.The gene encoding glutamatesemialdehyde aminotransferase is used as the high efficient screen marker since the offsprings plant without the marker produce albino seedlings.Uroporphyrinogen methyltransferase is a novel red fluorescent indicators in bacteria,fungi and mammalian cells,but its application in plants is needed to further study.Tobacco is widely used as the model for chloroplast transformation due to high efficiency.In this study,we constructed the chloroplast transformation vector in which hemL,the product of glutamatesemialdehyde aminotransferase from Escherchia coli,is applied as the screen marker,and cobA,the product of uroporphyrinogen methyltransferase from maize as the fluorescent marker. Construction of tobacco chloroplast genetic transformation of the expression vector with the homologous recombination fragment trnI/trnA.The results are as follows:1.The tobacco chloroplast genome were extracted from the leaves using the improved high salt and low pH method,the genome size of 23 kb,OD₂₆₀:OD₂₈₀ in the value of 1.8 to 2.0 range,extracting the high quanlity chloroplast genome.2.According to published tobacco NCBI homologous recombination protein trnI/trnA,a modified plastid ribosomal RNA operon promoter Prrn,the 3'legion of the plastid psbA gene psbA3',useing tobacco chloroplast genome as a template,using PCR amplification trnI/trnA,Prrn,psbA3' the specific DNA fragment,amplified fragment sequencing results:trnI gene length of 969 bp,trnA gene length of 883 bp,Prrn gene length of 144 bp,psbA3' gene length of 508 bp,They display high homologous to the published sequences.3.According to published hemL and cobA sequence,respectively,useing glutamatesemialdehyde aminotransferase gene recombinant GAST from E.coli and uroporphyrinogen methyltransferase gene recombinant SUMT from maize as templates, PCR amplified hemL and cobA specific gene fragment.Fragment sequencing results: hemL gene length of 1288 bp,cobA gene length of 846 bp.They display 100%high homologous to the published sequences. 4.Respectively with the corresponding restriction enzyme to digest the corresponding recombinant,recycling,source fragments,according to pBluescript SK + multiple cloning sites and order,the difference between foreign clips were inserted into pBluescript SK +,construct a tobacco Novel chloroplast expression vector pBSK-TPTCPhemL,sequences as follows:trnI→Prrn→RBS→hemL→RBS→cobA→psbA3'→trnA.Hindâ…¢digestion with pBSK-TPTCPhemL and display the size of 7000 bp by agarose gel electrophoresis testing.To sum up,it has been successfully cloned coding tobacco chloroplast expression vector of gene fragments,and construct a tobacco expression vector.The constructed vector for the tobacco chloroplast transformation will facilitate the study of hemL and cobA as the screen and red fluorescent marker respectively,a highly effective security system and the transformation of the chloroplast bioreactor technology.This improvement in crop breeding has broad prospects.