RESUMO
Background: Bacterial wilt caused by Ralstonia solanacearum is the most devastating disease in peanut. Planting resistant peanut cultivars is deemed as the sole economically viable means for effective control of the disease. To understand the molecular mechanism underlying resistance and facilitate breeding process, differences in gene expression between seeds of Rihua 1 (a Virginia type peanut variety resistant to bacterial wilt) inoculated with the bacterial pathogen suspension (10(9) cfu ml-1) and seeds of the same cultivar treated with water (control), were studied using the GenefishingTM technology. Results: A total of 25 differentially expressed genes were isolated. Expression of genes encoding cyclophilin and ADP-ribosylation factor, respectively, were further studied by real time RT-PCR, and full length cDNAs of both genes were obtained by rapid amplification of cDNA ends. Conclusions: The study provided candidate genes potentially useful for breeding peanut cultivars with both high yield and bacterial wilt resistance, although confirmation of their functions through transgenic studies is still needed.
Assuntos
Arachis/genética , Fatores de Ribosilação do ADP/genética , Ralstonia solanacearum/patogenicidade , Imunidade Inata , Reação em Cadeia da Polimerase em Tempo Real , Análise de SequênciaRESUMO
To isolate differentially expressed peanut genes responsive to chilling, a suppression subtractive hybridization (SSH) cDNA library was constructed for a chilling tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2ºC and 15ºC, respectively, for 24 hrs. A total of 466 cDNA clones were sequenced, from which 193 unique transcripts (73 contigs and 120 singlets) were assembled. Of these unique transcripts, 132 (68.4 percent) were significantly similar to the sequences in GenBank non-redundant (nr) protein database, which belonged to diverse functional categories including metabolism, signal transduction, stress response, cell defense and transcriptional regulation. The remaining 61 (31.6 percent) showed no similarity to either hypothetical or known proteins. Six differentially expressed transcripts were further confirmed with real-time quantitative PCR (RT-qPCR).
Assuntos
Arachis/genética , Arachis/metabolismo , Temperatura Baixa , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Biblioteca Gênica , Transcrição GênicaRESUMO
Screening of peanut seeds resulting from 0.39 percent sodium azide treatment with NIRS calibration equation for bulk seed samples identified a plant with more than 60 percent oleate. Oleate content in individual seeds of the plant, as predicted by NIRS calibration equation for intact single peanut seeds, ranged from 50.05 percent ~ 68.69 percent. Three seeds with >60 percent oleate thus identified were further confirmed by gas chromatography. Multiple sequence alignments of the FAD2B gene from Huayu 22 (wild type) and peanut seeds with elevated oleate (mutant type) revealed a C281T transition in the coding region causing an I94T substitution in the oleoyl-PC desaturase, which may be responsible for reduction in the enzyme activity.
Assuntos
Ácido Oleico/metabolismo , Arachis/genética , Arachis/metabolismo , Agricultura , Ácidos Graxos Dessaturases/genética , Arachis/enzimologia , Azida Sódica/farmacologia , Sequência de Bases , Cromatografia Gasosa , Clonagem Molecular , Genes de Plantas/genética , Mutagênese , Sementes , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
A novel hybrid identification protocol was developed for F0:1 peanut seeds resulting from crosses between normal oleate cultivars with wild type FAD2B gene and high oleate genotypes with an A insertion in FAD2B gene. Presence of a series of overlapped peaks in trace file of the PCR product amplified with bF19/R1 primers was an indication of hybridity. This protocol may facilitate high oleate breeding and genetic studies in peanut.
Assuntos
Arachis/genética , Hibridização Genética , Reação em Cadeia da Polimerase , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
An efficient DNA extraction method was developed for peanut seed, where only 3-5 mg cotyledonary tissue was enough for more than 50 PCR reactions with a reaction volume of 15 ul. Both low copy number and high copy number DNA sequences were successfully amplified. Processing one seed sample only took about half an hour. Sampling had no significant effects on germination and development. The DNA extraction method makes it possible to identify transformants and conduct molecular marker studies prior to sowing, and thus may greatly hasten research progress.
Assuntos
DNA de Plantas/genética , Arachis/genética , Cotilédone/genética , DNA , Reação em Cadeia da Polimerase , Sementes/genética , Biotecnologia/métodos , Marcadores GenéticosRESUMO
This paper describes a simple, low cost and reliable DNA template preparation protocol for polymerase chain reaction (PCR) using immature leaves from peanut seeds or leaves from field-grown plants. The technique may find wide utility in studies involving PCR-based molecular markers, rapid screening for transformants and gene cloning.