[show abstract][hide abstract] ABSTRACT: In this study, the aroA-M12 gene encoding bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and the Btslm recombinant gene encoding Bacillus thuringiensis (Bt) toxin gene were introduced into a Brassica napus variety, Xiangyou No. 15, via Agrobacterium-mediated transformation using glyphosate as a selectable agent. PCR amplification and Southern blot analyses of T0 transgenic plants showed that the alien genes were transferred and integrated stably into the genome of Xiangyou No. 15. Western blot further confirmed that the alien genes were expressed in these plants. Transgenic plants with tolerance to both glyphosate and the tested pest were demonstrated by bioassays. Our result also clearly shows that the aroA-M12 gene can be used as an efficient selectable marker gene instead of antibiotic resistant markers in plant transformation.
[show abstract][hide abstract] ABSTRACT: A promoter of the gene encoding beta-1, 3-glucanase isoenzyme GIII was amplified from barley genomic DNA using PCR. The GIII gene promoter, designated P(GIII), was ligated upstream of the gus report gene and pGIII-gus fusion fragment was then cloned into a binary vector pCAMBIA1300 for Agrobacterium-mediated transformation of rice (Oryza sativa L. cv. Taipei 309). PCR analyses indicated that the fusion gene was present in all T0 transgenic plants. The integration of the gene into rice genomic DNA was further confirmed by Southern blot. Histochemical staining and spectrofluorophotometric analyses of transgenic rice leaves showed that the GUS activity was increased after treatment with SA and fungal elicitor. Northern blot analysis also showed expression of such induction. Histochemical staining of T(1) rice seeds displayed marked GUS activity after induction with SA and elicitor, while no GUS activity was detected in untreated T(1) rice seeds. The results presented here strongly suggested that P(GIII) could be pathogen-inducible promoter.
Zhi wu sheng li yu fen zi sheng wu xue xue bao = Journal of plant physiology and molecular biology 05/2005; 31(2):143-8.
[show abstract][hide abstract] ABSTRACT: A mutant, aroAM12, exhibiting resistance to glyphosate produced in a previous study using the staggered extension process with aroA genes from Salmonella typhimurium and Eschrichia coli. In this paper, we constructed a vector pGRA1300 carrying aroAM12 gene, comprising transit peptide of Arabidopsis EPSPS, under the control of the CaMV35S promoter and used as selectable marker for cotton plant (Gossypium hirsutum L.) transformation. Transgenic cottons with increased resistance to glyphosate were obtained by cotransformtion of hypocotyl segments with Agrobacterium tumefaciens and selected directly on medium containing glyphosate. Regeneration of glyphosate-resistant calli was carried out on a MS basic medium containing 2,4-D 0.1 mg/L, KT 0.1 mg/L, cefotaxime 500 mg/L and glyphosate 60 micromol/L. Globular embryos were induced and then developed by culturing on MSB (MS salts+B(5) vitamins) medium supplemented with asparagine 1 g/L and glutamine 2 g/L, but not containing hormone, for 40 d. The developed plantlets were then removed and cultured on an MS medium. After about 20 d, the deeply-rooted shoots were in soil. PCR analysis showed that the aroAM12 gene was present in all T(0) transgenic plants. The integration of the aroAM12 gene in the genomic DNA of cotton was further confirmed by Southern blot, which showed that the transgenic plants carried one or two copies of the aroAM12 genes. Western blot analysis showed that a 48-kD band of was detected in all T(0) transgenic plants. There was no apparent corelation between copy numbers and the expression level of the aroAM12 gene. Greenhouse screening for glyphosate resistance was performed to test 65 independent T(0) plants by spraying (three times) with an aqueous suspension at a dose corresponding to 9.317 kg/ha of Roundup (once every 5 d). After 15 d, phenotype examination was carried out of the plants in comparison with untransformed control plants. Under these conditions, it was observed that the plants transformed with pGRA1300 showed high resistance to glyphosate whereas the control plants were all killed. The glyphosate resistance of T(1) generation was measured by spraying with Roundup, the numbers of glyphosate resistance and sensitive phenotypes showed Mendelian segregation ratio.
Zhi wu sheng li yu fen zi sheng wu xue xue bao = Journal of plant physiology and molecular biology 05/2004; 30(2):173-8.
[show abstract][hide abstract] ABSTRACT: A binary plant expression vector, pCM12-slm, carrying the aroAM12 mutant gene encoding bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and the Bts1m recombinant gene consisting of 331 N-terminal amino acids of CryIAc and 284 C-terminal amino acids of CryIAb has been constructed. The truncated Bts1 gene was fused with the PR1b signal peptide sequence and expressed in tobacco plants under the control of 2E-CaMV35S promoter and the omega (omega) translation enhancer sequence from tobacco mosaic virus. The mutant aroAM12 was fused with the transit sequence of tobacco EPSPS and expressed in tobacco plants under the control of the CaMV35S promoter. Tobacco leaves were transformed with Agrobacterium tumefaciens LBA4404 harboring the pCM12-slm plasmid, and the transgenic plants were selected directly on medium containing the herbicide. Forty glyphosate resistant plants were regenerated, with a transformation frequency of 27%. Transgenic plants were initially assessed for glyphosate resistance by placing leaf discs on shoot induction media containing the herbicide. Rooted plantlets, propagated from selected transgenic tobacco, were transferred to soil in a greenhouse and tested for glyphosate resistance by spraying them with Roundup at a commercial recommended dose. The glyphosate resistance assay indicated that all the transgenic plants showed highly resistant to the herbicide. The PCR assay showed that the aroAM12 gene was present in all of the 40 T0 transfer plants, and Bts1m genes present in 28 of 40 of the transgenic plants. Southern blot analysis further confirmed that the copy number of the transgenes varied from one to three copies in different transgenic plants. Northern blot and immunodot blot showed that the aroAM12 and Bts1m genes were expressed at the transcription and translation levels. Transgenic plants containing both the aroA M12 and Bts1m genes were further assessed for insect resistance. Tobacco leaves of T0 transgenic plants were infested with tobacco bollworm H. assulta larvae for 6 days. The result (table 1) showed that the survival rate of insect larvae was between 0-10%, and the growth of insect larvae was seriously inhibited, suggesting pCM12-slm as a dual functional vector with potential application in breeding of glyphosate and insect resistance transgenic plants.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 10/2003; 19(5):545-50.