Cloning EPSP Synthase Gene CDNA Of Allium Macrostemon Bunge And The Preliminary Studies On Its Function

5-enolpyruvlshimimate-3-phosphate synthase (EPSP synthase) is a key enzymethat is existed in prokaryote, yeasts, fungi, apicomplexan parasites, plants and algae incatalyzing the synthesize of aromatic amino acids. As a necessary key enzyme of theshikimate pathway, EPSPs convert phosphoenolpyruvate and shikimate 3-phophate to5-enolpyruvlshimimate-3-phosphate and phosphate..Glyphosate is a foliar applied,broad spectrum, low-toxicity organic phosphorous herbicide capable of controllingannual and perennial grasses and dicotyledonous weeds. Due to the similaritystructure between glyphosate and phosphoenolpyruvate, it can inhibit EPSP synthasepeculiarly by rivalrously integrated to EPSP synthase with phosphoenolpyruvate.Plants can obtain high doses glyphosate tolerance if the enzyme of this gene isoverproduced and accumulated or herbicide-insensitive enzymes produced due tosome amino acid mutation in active sites. Some bacterial herbicide-insensitive EPSPsgenes are cloned and transformed into several crops like soy bean and oil rape. Thetransgenic herbicide tolerance crops are widely planted now.Based on the homology alignment to other higher plants EPSP synthase gene,some conservative sequences are identify and a pair of degenerate oligonucleotideprimers designed. Total RNA ofAllium macrostemon Bunge is isolated and subject toRT-PCR. A 0.5 Kb amplified fragment is cloned and sequenced. Then RACE arecarried outo to clone the 3' and 5' end of the cDNA respectively. A 1821 bp cDNAcompleted sequence. It can encode a 522 amino acid deduced protein. By BLASTingthe database and analyzing the deduced protein structure, It is confirmed that thecDNA is for Allium macrostemon Bunge EPSP synthase. The cDNA is namedEPSPsA and was submitted to GenBank with the submitting No. : DQ462442.Semi-quantitative RT-PCR is carried out to investigate the EPSPsA geneexpression model of Allium macrostemon Bunge in different tissues with 18S rRNAas an internal control and EPSPsA 3'non-conserved region as target. A specific andstable RT-PCR semi-quantitative detection system established. The results show theEPSPsA are expressed in all the tissues tested in young leaves, roots, stems and old leaves in Allium macrostemon Bunge with an expression pattern that youngleaves＞roots＞stems＞old leaves, the relative expression levels are: 0.631,0.246,0.218,0.120 respectivly.Prokaryotic expression systems are applied for EPSPsA expression and to test itsglyphosate resistance. EPSPs cDNA with the contact signal sequence is cloned intopMAL-p2X vector, pMAL-p2X fusion expression vector pMAL-p2X-EPSPsA isconstructed and transferred into E. coli strain TB1. A predicted polypeptide areproduced by IPTG induction and SDS-PAGE analysis. And the improved glyphosateresistance are test when screen the bacteria on glyphosate. A truncated cDNA that getrid of some of the signal sequence is cloned into pREST-A vector and induced toexpress in E. coli BL21(DE3). And the bacteria are subject to glyphosate selection,.The glyphosate tolerance of the induced bacteria is apparently increased. It is thoughtthat the signal sequence may lead to dyn-location of the protein in bacteria.The cloned EPSPsA gene cDNA of Allium macrostemon Bunge, is also clonedinto Ti plasmid and the gene is under the control of constant promoter 35S to get aover-expression recombinant plant transform gene.. The recombinant are transformedinto a model plants-tobacco "WS38" by Agrobacterium tumefaciens mediatedtransformation. Hygromycin resistance callus are screened out. The callus arepropagated 3 weeks and then subjected to glyphosate resistance test. The transformedcallus are growing well in 800mg/L glyphosate medium while the non-transformedcontrol are very sensitive to glyphosate and putrescence in 50mg/L glyphosatemedium within 2w. It is further confirmed that the EPSPsA of Allium macrostemonBunge are glyphosate resistance and the molecular mechanism of Alliummacrostemon Bunge are concluded. The EPSPs cDNA ofAllium macrostemon Bungemay voluable in crop herbicide genetic engineering.