RESUMEN
The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin ß-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the ß-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein ß-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean ß-subunit gene. The level of 7S protein ß-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.
Asunto(s)
Agrobacterium tumefaciens/genética , Antígenos de Plantas/genética , Globulinas/genética , Glycine max/genética , Interferencia de ARN , Proteínas de Almacenamiento de Semillas/genética , Proteínas de Soja/genética , Antígenos de Plantas/metabolismo , Cotiledón/citología , Cotiledón/genética , Cotiledón/metabolismo , Técnicas de Transferencia de Gen , Genoma de Planta , Globulinas/metabolismo , Recombinación Genética , Proteínas de Almacenamiento de Semillas/metabolismo , Proteínas de Soja/metabolismo , TransgenesRESUMEN
Plant traits are important indices for regulating and controlling yield ability in soybean varieties. It is important to comprehensively study the quantitative trait locus (QTL) mapping for soybean plant traits, cloning related genes, and marker assistant breeding. In this study, 236 F2 generation plants and a derivative group were constructed by using Jiyu50 and Jinong18, obtained from Jilin Province. A total of 102 simple sequence repeat markers were used to construct a genetic linkage map. With 2 years of molecular and phenotypic data, QTL analyses and mapping were conducted for soybean maturity, plant height, main stem node, main stem branch, seed weight per plant, and more. Five main plant traits were analyzed via inclusive composite interval mapping using QTL IciMapping v2.2. Using one-dimensional scanning, a total of 30 QTLs were detected and distributed across 1 (A1), 4 (C2), and 12 (G). There were 9 linkage groups, including 16 major QTLs. Using two-dimensional scanning, 7 pairs of epistatic QTL interactions for maturity and plant height were detected in the soybean.
Asunto(s)
Mapeo Cromosómico/métodos , Glycine max/genética , Sitios de Carácter Cuantitativo , Cromosomas de las Plantas/genética , ADN de Plantas/análisis , Ligamiento Genético , Hibridación Genética , Repeticiones de MicrosatéliteRESUMEN
The role of high mobility group box 1 (HMGB1) has been demonstrated in stroke and coronary artery disease but not in peripheral arterial occlusive disease (PAOD). The pathogenesis of HMGB1 in acute and chronic vascular injury is also not well understood. We hypothesized that HMGB1 induces inflammatory markers in diabetic PAOD patients. We studied 36 diabetic patients, including 29 patients with PAOD, who had undergone amputation for diabetic foot and 7 nondiabetic patients who had undergone amputation after traumatic injury. Expression of HMGB1 and inflammatory markers were quantified using immunohistochemical staining. Mitochondrial DNA copy number was quantified using real-time polymerase chain reaction. Compared with that in the traumatic amputation group, HMGB1 expression in vessels was significantly higher in the diabetes and diabetic PAOD groups. In all subjects, arterial stenosis grade was positively correlated with the expression levels of HMGB1, 8-hydroxyguanosine, malondialdehyde, vascular cell adhesion molecule 1, and inflammatory markers CD3, and CD68 in both the intima and the media of vessels. Furthermore, HMGB1 expression level was positively correlated with 8-hydroxyguanosine, vascular cell adhesion molecule 1, nuclear factor-kB, CD3, and CD68 expression. Within the PAOD subgroup, subjects with HMGB1 expression had higher expression of the autophagy marker LC3A/B and higher mitochondrial DNA copy number. HMGB1 may be an inflammatory mediator with roles in oxidative damage and proinflammatory and inflammatory processes in diabetic atherogenesis. Moreover, it may have dual effects by compensating for increased mitochondrial DNA copy number and increased autophagy marker expression.
Asunto(s)
Aterosclerosis/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Pie Diabético/metabolismo , Proteína HMGB1/metabolismo , Amputación Quirúrgica , Arteriopatías Oclusivas/genética , Arteriopatías Oclusivas/metabolismo , Aterosclerosis/genética , Biomarcadores , Pie Diabético/genética , Pie Diabético/cirugía , Expresión Génica , Proteína HMGB1/genética , Humanos , Inflamación , Estrés Oxidativo , Enfermedad Arterial Periférica/genética , Enfermedad Arterial Periférica/metabolismoRESUMEN
Seed number per pod is an important component of yield traits in soybean (Glycine max L.). In 2010, we identified a natural mutant with an increased number of four-seed pods from a soybean variety named 'Jinong 18' (JN18). Subsequent observations indicated that the trait was stably inherited. To identify and understand the function of genes associated with this mutant trait, we analyzed the genetic differences between the mutant (JN18MT01) and source variety (JN18) by transcriptome sequencing. Three types of tissues, axillary buds, unfertilized ovaries, and young pods at three different growth stages, V6, R1, and R3, were analyzed, respectively. The sequencing results yielded 55,582 expressed genes and 4183 differentially expressed genes (DEGs). Among these, the log2 ratio value of 162 DEGs was >10, and 13 DEGs had overlapping expression at three different growth stages. Comparisons of DEGs among three different growth stages yielded similar results in terms of the percentage of genes classified into each gene ontology (GO) category. DEGs were classified into 25 different functional groups in clusters of orthologous groups analysis. Proportions of the main functional genes differed significantly over developmental stages. A comparison of enriched pathways among the three developmental stages revealed that 646 unigenes were involved in 103 metabolic pathways. These results show that the development of four-seed pods is associated with a complex network involving multiple physiological and metabolic pathways. This study lays the foundation for further research on cloning and on the molecular regulation of genes related to the four-seed pod mutation.
Asunto(s)
Frutas/genética , Regulación de la Expresión Génica de las Plantas , Glycine max/genética , Proteínas de Plantas/genética , Carácter Cuantitativo Heredable , Semillas/genética , Transcriptoma , Frutas/anatomía & histología , Frutas/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Redes y Vías Metabólicas/genética , Anotación de Secuencia Molecular , Mutación , Fenotipo , Proteínas de Plantas/metabolismo , Semillas/crecimiento & desarrollo , Glycine max/anatomía & histología , Glycine max/crecimiento & desarrolloRESUMEN
Determining the insertion position of an exogenous gene in the target plant genome is one of the main issues in the transgenic plant field. This study introduced a simple, rapid, and accurate method to clone the flanking sequences of the transgenic bar gene as the anchoring gene in the transgenic maize genome using single-primer polymerase chain reaction (PCR). This method was based on the distribution of restriction sites in the maize genome and adopted the single-primer PCR method. Cloning the flanking sequences with the restriction site-anchored single-primer PCR simplified the experimental procedures by about 70% and reduced the experimental time by more than 80%. In conclusion, the restriction site-anchored single-primer PCR was a simple, rapid method to obtain the unknown flanking sequences in the transgenic plants.
Asunto(s)
Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa/métodos , Transgenes/genética , Zea mays/genética , Región de Flanqueo 5'/genética , Clonación Molecular , Cartilla de ADN , Genoma de PlantaRESUMEN
The insertion position of exogenous genes in plant genomes is usually identified by adapter ligation-mediated polymerase chain reaction (PCR), thermal asymmetric interlaced PCR, and restriction site extension PCR in transgenic plant research. However, these methods have various limitations, such as the complexity of designing primers and time-consuming and multiple-step procedures. The goal of this study was to establish an easier, more rapid, and more accurate method to clone flanking sequence using single-primer PCR in transgenic plants. Unknown flanking genome sequences in transgenic plants, including those in tobacco, soybean, rice, and maize, were cloned using the single-primer PCR method established in this study, with the Bar gene as the anchor gene. The primer 1 (P1), P2, and P3 PCRs obtained 4 sequences, and the completely correct flanking sequence of 508 bp that was obtained in the P3 PCR was verified by sequencing analysis. The single-primer PCR is more rapid and accurate than conventional methods, justifying its application widely in cloning flanking sequences in transgenic plants.
Asunto(s)
Clonación Molecular , Plantas Modificadas Genéticamente , Clonación Molecular/métodos , Genes de Plantas , Oryza/genética , Glycine max/genética , Zea mays/genéticaRESUMEN
In order to investigate the genetic characteristics of soybean Leguminivora glycinivorella resistance and to improve soybean resistance insectivorous breeding efficiency by applying the multi-generation joint analysis method of the major gene plus polygene model, 5 pedigrees and generations (P1, F1, P2, F2, and F2:3) were used as the materials to perform the soybean L. glycinivorella resistance multi-generation joint analysis. The results showed that soybean resistance to L. glycinivorella was controlled and inherited by an additive major gene plus additive, dominant polygene. The major gene had a negative additive effect (d = -0.1633). The combination of the anti-L. glycinivorella genes showed negative heterosis. Because the polygene additive effects were positive, the polygene effects would increase the insect herbivory rate in the F1 generation. This hybrid combination showed an insect herbivory rate polygenic heritability of 21.9556 and 54.3490% in the F2 and F2:3 pedigrees, which presented a high heritability. Therefore, it was appropriate to perform the selective breeding of the insect herbivory rate in the late generation.
Asunto(s)
Genes de Plantas , Glycine max/genética , Lepidópteros/fisiología , Modelos Genéticos , Herencia Multifactorial , Animales , Cruzamiento , Cruzamientos Genéticos , Glycine max/inmunología , Glycine max/parasitologíaRESUMEN
Hyperglycemia-induced reactive oxygen species production can cause diabetes and its complications, including atherosclerosis. The role of mitochondrial DNA variants and mitochondrial copy number in the pathogenesis of diabetic atherogenesis is not well understood. We examined 36 diabetic patients who had undergone amputation for diabetic foot and seven non-diabetic patients who had undergone amputation after traumatic injury. Mitochondrial DNA was extracted and used for sequencing. Single nucleotide polymorphisms (SNPs) relative to the Cambridge reference sequence were analyzed. Mitochondrial DNA copy number was quantified by real-time PCR. Twenty-one novel variants were detected in 29 diabetic patients with arterial stenosis; six of the variants were heteroplasmic, and most occurred in highly evolutionarily conserved residues. These variants were more prevalent in patients with arterial stenosis than in those without stenosis. The novel variants included four in complex I (ND1: C3477A/C, A3523A/G; ND5: C13028A/C, C13060A/C), one in complex IV (COX1: T6090A/T), and one in rRNA (12srRNA: G857G/T). Compared with non-diabetic patients, the diabetic patients had significantly less mitochondrial DNA. Furthermore, among diabetic patients with arterial stenosis, there was a significant positive correlation between mitochondrial DNA copy number and the number of total SNPs. In conclusion, we identified six novel heteroplasmic mitochondrial DNA variants among diabetic patients with arterial stenosis, and we found that diabetic atherogenesis is associated with decreased amounts of mitochondrial DNA.
Asunto(s)
Aterosclerosis/genética , Variaciones en el Número de Copia de ADN/genética , ADN Mitocondrial/genética , Complicaciones de la Diabetes/genética , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada/genética , Análisis Mutacional de ADN , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/genética , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/genética , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Polymerase chain reaction (PCR) is the foundation of SSR molecular marker technology. We used sib rice varieties J518, XD1 and SD23 as experimental materials, selecting 30 pairs of SSR primers, including RM127, RM337 and RM5172, covering the rice genome, and performed single- and double-SSR primer combined analyses. We found that under the same PCR system and conditions, a single primer of the SSR primer pairs could amplify the same fragments as double primers do. The sequencing results demonstrated that some amplified fragments that we previously believed to come from double primers were actually produced by a single primer. The use of this kind of primer, such as the RM127 primer pair, for marker-assisted breeding will therefore be misleading. Additionally, using the same PCR system and conditions, some single primers that are part of SSR primer pairs can amplify many more specific fragments than double-SSR primers. For instance, in the case of the RM5172 primer pair, a single primer P1 amplified approximately three times the number of fragments as the double primer. This information can contribute to research on genetic diversity of species, understanding of genetic relationships and identification of germplasm resources. Accordingly, combined analyses of single- and double-primer amplification products not only can remove single-primer amplification fragments and false-positives from double-primer amplification products in order to improve test accuracy, but also can facilitate research on genetic diversity, exploration of phylogenetic relationships and identification of germplasm resources. We define this method as "single- and double-SSR primer combined analyses".
Asunto(s)
Oryza/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Cartilla de ADN , Polimorfismo GenéticoRESUMEN
Polymerase chain reaction (PCR) provides a foundation for simple sequence repeat molecular marker-assisted selection (SSR MAS) in soybean. This PCR system and its various conditions have been optimized by many researchers. However, current research on the optimization of the PCR system focuses on double-primer PCR products. We compared single- and double-SSR primer PCR products from 50 soybean samples and found that the use of single-PCR primers in the reaction system can lead to amplified fragments of portions of the SSR primers in the PCR process, resulting in both false-positives and fragment impurity of double-primer PCR amplification, inconvenient for subsequent analysis. We used "single-primer PCR correction" to eliminate interference caused by single-primer nonspecific PCR amplification and improve PCR quality. Using this method, the precision and success rates of SSR MAS in soybean can be increased.
Asunto(s)
Contaminación de ADN , Cartilla de ADN/química , Marcadores Genéticos , Glycine max/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de BasesRESUMEN
Polymerase chain reaction (PCR) technology plays an important role in molecular biology research, but false-positive and nonspecific PCR amplification have plagued many researchers. Currently, research on the optimization of the PCR system focuses on double-primer-based PCR products. This research has shown that PCR amplification based on single-primer binding to the DNA template is an important contributing factor to obtaining false-positive results, fragment impurity, and nonspecific fragment amplification, when the PCR conditions are highly restricted during PCR-based target gene cloning, detection of transgenic plants, simple-sequence repeat marker-assisted selection, and mRNA differential display. Here, we compared single- and double-primer amplification and proposed "single-primer PCR correction"; improvements in PCR that eliminate interference caused by single-primer-based nonspecific PCR amplification were demonstrated and the precision and success rates of experiments were increased. Although for some kinds of experiments, the improvement effect of single-primer PCR correction was variable, the precision and success rate could be elevated at 12-50% in our experiment by this way.
Asunto(s)
Cartilla de ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Southern Blotting , Oryza/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
INTRODUCTION: Ischemia/reperfusion injury (IRI) represents the single major antigen-independent factor implicated in pathogenesis of chronic graft dysfunction. Tacrolimus is a calcineurin inhibitor, which has been suggested to be helpful in cyclosporine-related chronic toxicity. Rapamycin has antiproliferative properties that may impair renal regeneration after IRI. Therefore, immunosuppressive drugs might impair renal graft outcome in those organs suffering IRI. MATERIAL AND METHODS: C57B1/6 male mice subjected to 45 minutes of renal pedicle ligation were reperfused for 24 hours. Mice were treated with rapamycin, cyclosporine, or tacrolimus. Blood and renal tissue samples were collected at 24 hours after IRI. Urea levels were measured. Heme Oxygenase 1 (HO-1) gene transcript was amplified by a real-time polymerase chain reaction technique. RESULTS: Animals treated with cyclosporine and subjected to IRI showed impaired renal function that peaked at 24 hours. Additional pretreatment with rapamycin produced even more impairment of renal function, when compared with controls. However, tacrolimus pretreatment was associated with a better renal outcome. HO-1 expression was upregulated after IRI by 2.6 arbitrary units at 24 hours. Rapamycin showed worse impairment of renal function. CONCLUSION: Tacrolimus was not associated with worsening renal function when compared with animals just subjected to IRI. Upregulation of HO-1 may be an attractive approach to limit graft injury.