Cloning And Analysis Of CDNA Sequence Coding EPSP Synthase In Brachypodium Distachyon

The enzyme 5-enolpyruvylshikimate 3-phosphate synthase(EPSPs) is a critical enzyme in the shikimate pathway,which was present in bacteria,fungi,and plant.Interest in the characterization of plant EPSPS has been increased significantly since the enzyme was identified as the primary target of the broad-spectrum,nonselective herbicide glyphosate.This study was carried to obtain the EPSPs gene from Brachypodium distachyon,on the basis of the known EPSPs genes of plants,and the main results were following:1.By screening the EST database of B.distachyon,five sequences homologous to the EPSPs genes of rice and wheat were obtained and assembled to a 976 bp contig.This contig was 5' part of the EPSPs gene,containing a 827 bp partial ORF and 149 bp flanking sequence. A pair of PCR primers was then designed and the 5' part of the EPSPs gene was cloned and re-sequenced.2.Using 3' RACE,the 3' part of EPSPs gene of B.distachyon was obtained,it was 994 bp long,containing 774 bp coding sequence and 217 bp 3' non-translating region.The obtained 5' and 3' part of EPSPs were assembled to a 1899 bp Sequence,containing a 1530 bp ORF,a 149 bp 5' flanking sequence and 217 bp 3' non-translated region,and could be translated to 510 amino acids.Sequence comparison indicated that the coding sequences of EPSPs gene of B.distachyon are high similar to the known genes in plant. However,there is great difference in the 5' flanking and 3' non-translated sequences.3.Phylogenic analysis of the EPSPs in plants indicated the monocotyledon and dicotyledon plants could be clustered separatedly,and B.distachyon was more homologous to wheat and lolium than rice and Mays.Theses results suggested that EPSPs gene might be species specific.