RESUMO
Genotyping is a critical step for molecular marker-assisted selection in rice. Rice genomic DNA samples for genotyping are typically isolated from living tissues such as seedlings. This requires the germination of all candidate seeds and extraction of DNA from the seedlings. Currently, an ideal individual is selected from a very large number of plants, which is time- and labor-consuming, requiring several transplantations of materials and sampling processes. In this study, we developed a simplified genomic DNA extraction protocol in rice by using amylase to treat half-seeds. The yields of genomic DNA from a half-seed of Indica and Japonica rice were greater than 203.8 ± 32.5 and 143.2 ± 25.5 ng, respectively, and the 260/280 nm absorbance ratio was 1.75-2.10. The DNA was confirmed to be sufficient for polymerase chain reaction amplification and can be used in a marker-assisted selection program.
Assuntos
DNA de Plantas/isolamento & purificação , Genômica , Germinação/genética , Oryza/genética , DNA de Plantas/genética , Genoma de Planta , Genótipo , Plântula/genética , Sementes/genéticaRESUMO
A few insect control genes of Bacillus thuringiensis have been modified successfully to increase the expression in plants by replacing rare codons, increasing GC content, and avoiding the DNA elements that could cause premature transcription termination, mRNA instability, and potential methylation. However, the modification process was intricate and often confused researchers. In this study, we adopted a simple method to modify Cry1Ab only by individually replacing its amino acid sequence with corresponding rice-preferred codons based on analysis of 92,188 coding DNA sequences. Unexpectedly, all elements of A+T richness, which terminate or destabilize transcription in plants, were avoided in the newly designed mCry1Ab. However, mCry1Ab had 2 notable features: less synonymous codons and high GC content. mCry1Ab only employed 22 of the 61 codons to encode protein and had an enhanced GC content of 65%. The increase in GC content caused abundant potential methylation signals to emerge in mCry1Ab. To test whether mCry1Ab could be expressed in rice, we transferred it into Oryza japonica variety Wanjing97. Insect bioassays revealed that transgenic plants harboring this gene driven by 2 promoters, CaMV35S and OsTSP I, were highly resistant to rice leaffolder (Cnaphalocrocis medinalis). Analysis of R0 to R2 generation plants indicated that the mCry1Ab was inherited stably by the progeny. Our study provided a simple modified method for expressing exogenous genes in rice and confirmed that less synonymous codons and high GC content do not affect transgene expression in rice.
Assuntos
Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Oryza/genética , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Lepidópteros/patogenicidade , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras GenéticasRESUMO
The aim of this study was to develop an event-specific qualitative and real-time quantitative polymerase chain reaction (PCR) method for detection of herbicide-tolerance genetically modified (GM) soybean A2704-12. The event-specific PCR primers were designed, based on the 5'-flanking integration sequence in the soybean genome, to amplify the 239-bp target fragment. Employing the same event-specific primers, qualitative PCR and real-time quantitative PCR detection methods were successfully developed. The results showed that the A2704-12 event could be specifically distinguished from other GM soybean events. In the qualitative PCR assay, the limit of detection was 0.05%, and in the real-time quantitative PCR assay, the limit of detection was less than 0.01%. Moreover, our genomic DNA (gDNA) extraction protocol is high-throughput, safe, and low-cost. The event-specific PCR assay system is cost-efficient by using SYBR Green I in real-time PCR, and by using the same primers in both the qualitative and quantitative PCR assays. We therefore developed a high-throughput, low-cost, and event-specific qualitative and quantitative PCR detection method for GM soybean A2704-12. The method would be useful for market supervision and management of GM soybean A2704-12 due to its high specificity and sensitivity.
Assuntos
Glycine max/genética , Ensaios de Triagem em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Herbicidas/toxicidade , Ensaios de Triagem em Larga Escala/economia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Reação em Cadeia da Polimerase/economia , Glycine max/efeitos dos fármacos , Glycine max/crescimento & desenvolvimentoRESUMO
Low temperature is a major environmental stress in rice cultivating and production. The alternative oxidase 1 (AOX1) gene is potentially important for genetic engineering to increase cold adaptation. However, previous studies related to this effect have mostly focused on the dicot plants Arabidopsis and tobacco, whereas functional research on rice is limited. In this study, we cloned a rice predominant cold-response AOX1 gene, OsAOX1a. Transgenic rice plants with overexpression of OsAOX1a were obtained. We found that OsAOX1a overexpression could strongly enhance the cold growth of seedlings, especially with respect to root extension. However, growth between transgenic and control plants did not differ under normal conditions. Furthermore, the lipid peroxidation and ion leakage rate were determined after cold treatment in transgenic plants. Both factors were reduced by OsAOX1a overexpression, which revealed that OsAOX1a could reduce oxidative damage under cold stress. Taken together, our results suggested that overexpressing OsAOX1a could improve growth performance of rice under cold stress, which might be closely related to the reduction of reactive oxygen species generation and oxidative damage.
Assuntos
Temperatura Baixa , Proteínas Mitocondriais/metabolismo , Oryza/genética , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Transporte de Íons , Peroxidação de Lipídeos , Proteínas Mitocondriais/genética , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Oxirredutases/genética , Proteínas de Plantas/genéticaRESUMO
The isolation of high-quality genomic DNA (gDNA) is a crucial technique in plant molecular biology. The quality of gDNA determines the reliability of real-time polymerase chain reaction (PCR) analysis. In this paper, we reported a high-quality gDNA extraction protocol optimized for real-time PCR in a variety of plant species. Performed in a 96-well block, our protocol provides high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost-efficient than traditional DNA extraction methods. The method takes 10 mg leaf tissue to yield 5-10 µg high-quality gDNA. Spectral measurement and electrophoresis were used to demonstrate gDNA purity. The extracted DNA was qualified in a restriction enzyme digestion assay and conventional PCR. The real-time PCR amplification was sufficiently sensitive to detect gDNA at very low concentrations (3 pg/µL). The standard curve of gDNA dilutions from our phenol-chloroform-free protocol showed better linearity (R(2) = 0.9967) than the phenol-chloroform protocol (R(2) = 0.9876). The results indicate that the gDNA was of high quality and fit for real-time PCR. This safe, high-throughput plant gDNA extraction protocol could be used to isolate high-quality gDNA for real-time PCR and other downstream molecular applications.
Assuntos
DNA de Plantas/isolamento & purificação , Brassica napus , DNA de Plantas/química , Genoma de Planta , Oryza/química , Folhas de Planta/química , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Mapeamento por Restrição , Triticum/química , Zea mays/químicaRESUMO
Rice false smut (RFS) is an important rice disease that is caused by the pathogen Ustilaginoidea virens. In this study, we developed a real-time polymerase chain reaction (PCR) assay to detect U. virens and to estimate the level of disease. The genomic DNAs of U. virens and rice were extracted together from the rice samples. Real-time PCR assays were performed and compared to conventional nested-PCR assays. The real-time PCR assay presented a consistent linearity of the standard curve (R(2) = 0.9999). The detection limit could be as low as 40 fg U. virens DNA with a rice genomic DNA background on using the real-time PCR assay, which showed significantly higher sensitivity than the conventional nested-PCR assay. We conclude that the real-time PCR quantitative assay is a useful tool for detecting U. virens and for early defense and control of RFS.