玉米GSAT突变体作为筛选标记的植物表达载体构建.pdfVIP

Abstract Bacterial selectable marker genes conferring antibiotic and herbicide resistance play a vital role in plant genetic engineering. However, because of its potential ecological security and hazards to human health, the application of the marker gene has been controversial. In recent years, public concern and regulatory requirements have stimulated the development of alter-native selection systems. Studies have found, a mutated Synechococcus elongatus HemL encoding glutamate 1-semialdehyde aminotransferase (GSAT) is an efficient safty selectable marker gene in plant nuclear genome genetic transformation. In fact, GSAT is irreversibly inhibited by gabaculine (3-amino-2, 3-dihydrobenzoic acid), but the mutated enzyme is insensitive to gabaculine. Based on the previous study, we constructed chloroplast genome and nuclear 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 ，by PCR, this mutated gene successfully replaced pCAMBIA1301 GUS gene as a selectable marker gene, then the nuclear genetic transformation expression vector was constructed. 2. The tobacco chloroplast genome was extracted from the leaves using the modified high salt and low pH method. The fragments of trnA and trnI were amplified by PCR using tobacco chloroplast genome DNA as the template. Sequence analysis confirmed the homogeneity of the amplified fragments were up to 99% comparing with the published sequences. 3. To constratuct the chloroplast transformation vectors, we amiplified the related gene fragments by overlapping PCR and inserted into pUC119 pl