RESUMEN
The xylA gene, encoding xylose isomerase, was cloned as a 1342-bp BamHI/SacI fragment from the E. coli. As a selection marker, the xylA gene was fused between the enhanced CaMV 35S promoter (E35S) and terminator (35St) in pBAC413 (Fig.2). pBAC413 was constructed to prevent the expression of sbeIIb in maize. PDS1000/He was used to bombard maize calli, which were induced to form by the elite inbred lines. The selection was carried out on the media containing concentrations of xylose from 0 to 100%. The results showed that the media containing 50% to 100% D-xylose were better, but differed with the genotype of maize (Tables 1 and 2). Successful integration of xylA gene into the maize genome was confirmed by DNA dot blotting, PCR and PCR-Southern hybridization (Figs.4 to 6). A method was established in which transformed maize cells were successively screened on a medium containing xylose instead of antibiotic and herbicide for bio-safety.
Asunto(s)
Isomerasas Aldosa-Cetosa/genética , Zea mays/genética , Southern Blotting , Genoma de Planta/genética , Immunoblotting , Modelos Genéticos , Reacción en Cadena de la Polimerasa , Transformación Genética/genéticaRESUMEN
Expression vector pBAC128F, which carries DREB transcriptional factor gene driven by drought inducing promoter rd29B and bar gene driven by CaMV 35S promoter and maize Adh1 gene first intron, was transferred into the explants of immature inflorescence and immature embryos of hexaploid winter wheat cv. 8901, 5-98, 99-92 and 104 by particle bombardment. More than 70 resistant transgenic plants were obtained. Genomic PCR and RNA dot blotting analyses showed that DREB gene had been integrated into wheat genome of the transgenic plants (T0 and T1) and was well expressed in offspring seed of different transgenic lines. The content of proline in leaves and seeds of T2 transgenic lines was analyzed. Among 16 tested transgenic lines, 10 transgenic lines exhibited more than two fold of proline level in leaves as compared with CK plants. Under drought condition, after stopping water for 15 days the leaves of transgenic lines were still green, while CK were faded. After rewatering for 10 days, the leaves of transgenic lines maintained their green, while all CK plants were dead. Our research suggested that introducing a novel DREB transcriptional factor into wheat is an effective way to improve its drought-tolerance ability.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Sequías , Factores de Transcripción/fisiología , Agua/metabolismo , Ácido Abscísico/farmacología , Comunicación Celular , Desecación , Expresión Génica , Regulación de la Expresión Génica de las Plantas/fisiología , Plantas Modificadas Genéticamente , Plantones , Transcripción Genética/fisiología , TriticumRESUMEN
Fourteen wheat cultivars were identified into six types of Wx proteins combinations using 6% SDS-PAGE. PCR primers were designed according to the three Wx genes sequences and their mutants, respectively. A 327 bp-band was amplified from the Wx-A1 mutant,while the band was absent for the normal alleles at the Wx-A1 locus,as well as the presence or absence of a 187 bp PCR fragment at the Wx-B1 locus and a 700 bp PCR fragment at the Wx-D1 locus, respectively, corresponding to the normal and mutant alleles. Compared with the former studies, shorter and more different PCR products at three loci, amplified by the primers designed for Wx-B1 gene can be separated in 2% agarose gel, which enables screening breeding lines for noodle use faster and effectively.
Asunto(s)
Polimorfismo Genético , Almidón Sintasa/genética , Triticum/genética , Electroforesis en Gel de Poliacrilamida , Reacción en Cadena de la Polimerasa , Triticum/enzimologíaRESUMEN
PCR is a powerful tool for the amplification of genetic sequences. It has been widely applied in molecular biology. It is generally used to amplify short segments (several hundreds basepairs to several kilobasepairs). It is difficult to amplify a long DNA segment. Based on the sequenced genes, it is known that most intact genes are very long. And intact gene is very important for the gene to express specially and effectively. Long PCR is a very useful tool to amplify intact genes for constructing special expression vectors. We have tried several chemicals to optimize long PCR system and found betaine was the best. Betaine, as an amino acid analogue with small tetraalkylammonium ions, could remarkably improve the amplification of long targets from the plant genome. The suitable concentration of betaine was between 1.0 mol/L and 2.5mol/L. We could effectively amplify a 9 kb DNA segment from maize genome DNA and a 16 kb DNA segment from plasmid. It was shown that different primers and different targets (different GC content) needed different concentrations of betaine. Betaine can reduce or eliminate non-special amplification. In the meantime we tried other additive chemicals, such as DMSO, glycerin, formamide. They were no notable results in long PCR.
Asunto(s)
Betaína/farmacología , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Simple sequence repeat (SSR) molecular marker was used to measure the genetic diversity of D-genome in 26 synthesized hexaploid wheat (AABBDD) introduced from CIMMYT. Twenty-three D-genome specific SSR primers were selected for PCR amplification, among which 22 primers can detect polymorphism. A total of 92 alleles were identified at 23 loci using the above SSR primers, with an average of 4 alleles per locus. The 92 alleles were used to calculate Nei's similarity index (GS) and the genetic distance (GD). It was also found that the mean genetic distance between 26 synthesized hexaploid wheat was 0.4955, which was obviously high. From the above results, it can be indicated that the genetic variation of D-genome in synthesized hexaploid wheat was abundant and could be used to improve the genetic diversity in wheat breeding. Interestingly, synthesized hexaploid wheat 17 and 18 shared the same D-genome donor, but three of 23 detected SSR loci were polymorphic between the two materials. Therefore, during the period of allopolyploidization, there was genetic differentiation in repeat region of donor genome.