RESUMO
Moth bean is the most drought and heat tolerant cultigens among Asian Vigna. We performed comparative transcriptome analysis of moth bean cultivar "Marumoth" under control and stress condition. De novo transcriptome assembly was carried out by using Velvet followed by Oases softwares. Differential expression analyses, SSR identification and validation and mapping of pathways and transcription factors were conducted. A total of 179,979 and 201,888 reads were generated on Roche 454 platform and 48,617,205 and 45,449,053 reads were generated on ABI Solid platform for the control and stressed samples. Combined assembly from Roche and ABI Solid platforms generated 16,090 and 15,096 transcripts for control and stressed samples. We found 1287 SSRs and 5606 transcripts involved in 179 pathways. The 55 transcription factor families represented 19.42% of total mothbean transcripts. In expression profiling, ten transcripts were found to be up-regulated and 41 down-regulated while 490 showed no major change under moisture stress condition. Stress inducible genes like Catalase, Cyt P450 monooxygenase, heat shock proteins (HSP 90 and HSP 70), oxidoreductase, protein kinases, dehydration responsive protein (DRP), universal stress protein and ferridoxin NADH oxidoreductase genes were up-regulated in stressed sample. Genes which might be involved in moisture stress tolerance in moth bean were identified and these might be useful for stress tolerance breeding in moth bean and other related crops.
RESUMO
Newcastle disease (ND) is highly contagious, economically important viral disease affecting most of avian species worldwide. Newcastle disease virus (NDV) has single stranded negative sense RNA genome which encodes for six structural and two non-structural proteins. Envelope glycoproteins i.e. hemagglutinin-neuraminidase (HN) and the fusion (F), elicit protective immune response. In this study, HN and F genes of velogenic (virulent) strain were amplified and cloned at multiple cloning sites A and B, respectively into pIRES bicistronic vector for use as bivalent DNA vaccine against ND. The recombinant plasmid was characterized for its orientation by restriction enzyme digestion and PCR. Expression of HN and F genes was assessed in transfected Vero cells at RNA level using RT-PCR in total RNA as well as protein level using IFAT, IPT and western blot using NDV specific antiserum. All these experiments confirmed that HN and F genes cloned in recombinant pIRES.nd.hn.f are functionally active. The recombinant construct is being evaluated as DNA vaccine against ND.
Assuntos
Clonagem Molecular , Proteína HN/genética , Vírus da Doença de Newcastle/genética , Vacinas de DNA/genética , Proteínas Virais de Fusão/genética , Animais , Chlorocebus aethiops , DNA Viral/genética , Vírus da Doença de Newcastle/imunologia , Proteínas Recombinantes de Fusão/genética , Transfecção , Vacinas de DNA/imunologia , Células Vero , Proteínas Virais de Fusão/imunologiaRESUMO
Granulocyte-macrophage colony stimulating factor (GMCSF), a multifunctional cytokine can enhance immune responses when administered along with DNA vaccine. Aim of the present study was to clone and express the chicken GMCSF cytokine for use as 'genetic adjuvant'. Chicken GMCSF gene 435bp was amplified using specific primers in which restriction sites of BamHI and HindIII were at forward and reverse primers respectively. The PCR product was cloned into eukaryotic expression vector pcDNA 3.1(+) and clones were confirmed by restriction digestion and nucleotide sequencing. Functional activity of recombinant GMCSF was checked by expression of GMCSF specific mRNA in transfected Vero cells by RT-PCR of total RNA isolated from transfected Vero cells. The recombinant plasmid can be used as genetic adjuvant in chicken.
Assuntos
Clonagem Molecular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Sequência de Bases , Galinhas , Chlorocebus aethiops , Vetores Genéticos , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Células VeroRESUMO
A simple and efficient protocol for recurrent somatic embryogenesis and plant regeneration is one of the prerequisites for genetic improvement of guava. An efficient reproducible regeneration somatic embryogenesis protocol was developed in four genotypes of Psidium guajava L. using immature zygotic embryo as starter explant. Somatic embryogenesis induction was obtained on MS basal medium supplemented with 2.0 mgL-1 2, 4-D, 400 mgL-1 L-glutamine, 6% sucrose and 500 mgL-1 Malt extract. Following SE induction different developmental stages of somatic embryos (Globular, heart-shaped, torpedo, cotyledonary) was directly obtained and further recurrent embryogenesis also obtained upon prolonged incubation in induction media. Addition of polyethylene glycol (50 mgL-1) significantly improved the embryos maturation in MS supplemented with and 3% sucrose. The regeneration in MS medium supplemented with BAP (0.5 mgL-1), NAA (0.2 mgL-1), casein hydrolysate (100 mgL-1) and 3% sucrose. High plant regeneration frequency and intensity of somatic embryos (58.5%) obtained. Plant maturation on MS medium supplemented with BAP 2.0 mgL-1 with 2% sucrose. The rooted plants was successfully acclimatize in the greenhouse with a survival rate of 85%. This somatic embryogenesis protocol developed would be helpful in establishment of genetically modified guava aimed at seedlessness, increased shelf life and wilt disease.