Construction Of The Vectors For Expressing A Maize GSAT Mutant As A Selectable Marker In Plant

Bacterial selectable marker genes conferring antibiotic and herbicide resistanceplay a vital role in plant genetic engineering. However, because of its potentialecological security and hazards to human health, the application of the marker genehas been controversial. In recent years, public concern and regulatory requirementshave stimulated the development of alter-native selection systems. Studies have found,a mutated Synechococcus elongatus HemL encoding glutamate1-semialdehydeaminotransferase (GSAT) is an efficient safty selectable marker gene in plant nucleargenome genetic transformation. In fact, GSAT is irreversibly inhibited by gabaculine(3-amino-2,3-dihydrobenzoic acid), but the mutated enzyme is insensitive togabaculine. Based on the previous study, we constructed chloroplast genome andnuclear genome expression vectors using maize GSAT mutant as a selectable marker.The results are as follows:1. According to sequence analysis of the mutant maize GSAT gene mGSATM，byPCR, this mutated gene successfully replaced pCAMBIA1301GUS gene as aselectable marker gene, then the nuclear genetic transformation expression vectorwas constructed.2. The tobacco chloroplast genome was extracted from the leaves using the modifiedhigh salt and low pH method. The fragments of trnA and trnI were amplified byPCR using tobacco chloroplast genome DNA as the template. Sequence analysisconfirmed the homogeneity of the amplified fragments were up to99%comparingwith the published sequences.3. To constratuct the chloroplast transformation vectors, we amiplified the relatedgene fragments by overlapping PCR and inserted into pUC119plasmid. Thecommonly used selection marker gene aadA was used as the control andexpressed under the promoter prrn, as well as mGSATM with the followingterminator psbA. The species-specific chloroplast flanking sequences (trnA andtrnI) inserted at the endis of two cistron. The final expression element was trnI-prrn-aadA-prrn-gsatm-psbA-trnA.4. Using the mGSATM gene as a selectable marker, we transformed it to tobacco byagrobacterium-mediated transformation, the transformants were selected by50mg/L Hyg, and transgenic plants were obtained.In general, we constructed two vectors for a nuclear and chloroplasttransformation using the mGSATM gene as the selection marker. The constructedtobacco chloroplast expression vector using the mGSATM gene as the selectablemarker provided the feasible application of this marker.