RESUMEN
OBJECTIVE: For efficient biofarming we attempted to enrich plant interstitial fluid (IF)/apoplastic fluid with targeted recombinant therapeutic protein. We employed a synthetic human Glucocerebrosidase (GCB), a model biopharmaceutical protein gene in this study. RESULTS: Twenty one Nicotiana varieties, species and hybrids were initially screened for individual IF recovery and based on the findings, we selected Nicotiana tabacum NN (S-9-6), Nicotiana tabacum nn (S-9-7) and Nicotiana benthamiana (S-6-6) as model plants for raising transgenic expressing GCB via Agrobacterium mediated transformation under the control of M24 promoter; GCB specific activity in each transgenic lines were analyzed and we observed higher concentration of recombinant GCB in IF of these transgenic lines (S-9-6, S-9-7, and S-6-6) in comparison to their concentration in crude leaf extracts. CONCLUSION: Recovery of valuable therapeutics in plant IF as shown in the present study holds great promise for promoting plant based biofarming. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:726-736, 2017.
Asunto(s)
Glucosilceramidasa/metabolismo , Extractos Vegetales/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Glucosilceramidasa/genética , Humanos , Extractos Vegetales/genética , Hojas de la Planta/química , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismoRESUMEN
MAIN CONCLUSION: We have designed two near- constitutive and stress-inducible promoters (CmYLCV9.11 and CmYLCV4); those are highly efficient in both dicot and monocot plants and have prospective to substitute the CaMV 35S promoter. We performed structural and functional studies of the full-length transcript promoter from Cestrum yellow leaf curling virus (CmYLCV) employing promoter/leader deletion and activating cis-sequence analysis. We designed a 465-bp long CmYLCV9.11 promoter fragment (-329 to +137 from transcription start site) that showed enhanced promoter activity and was highly responsive to both biotic and abiotic stresses. The CmYLCV9.11 promoter was about 28-fold stronger than the CaMV35S promoter in transient and stable transgenic assays using ß-glucuronidase (GUS) reporter gene. The CmYLCV9.11 promoter also demonstrated stronger activity than the previously reported CmYLCV promoter fragments, CmpC (-341 to +5) and CmpS (-349 to +59) in transient systems like maize protoplasts and onion epidermal cells as well as transgenic systems. A good correlation between CmYLCV9.11 promoter-driven GUS-accumulation/enzymatic activities with corresponding uidA-mRNA level in transgenic tobacco plants was shown. Histochemical (X-Gluc) staining of transgenic seedlings, root and floral parts expressing the GUS under the control of CmYLCV9.11, CaMV35S, CmpC and CmpS promoters also support the above findings. The CmYLCV9.11 promoter is a constitutive promoter and the expression level in tissues of transgenic tobacco plants was in the following order: root > leaf > stem. The tobacco transcription factor TGA1a was found to bind strongly to the CmYLCV9.11 promoter region, as shown by Gel-shift assay and South-Western blot analysis. In addition, the CmYLCV9.11 promoter was regulated by a number of abiotic and biotic stresses as studied in transgenic Arabidopsis and tobacco plants. The newly derived CmYLCV9.11 promoter is an efficient tool for biotechnological applications.
Asunto(s)
Arabidopsis/genética , Caulimovirus/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regiones Promotoras Genéticas/genética , Arabidopsis/fisiología , Flores/genética , Flores/fisiología , Expresión Génica , Genes Reporteros , Cebollas/genética , Cebollas/fisiología , Enfermedades de las Plantas/inmunología , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Protoplastos , Proteínas Recombinantes , Plantones/genética , Plantones/fisiología , Estrés Fisiológico , Nicotiana/genética , Nicotiana/fisiologíaRESUMEN
We have developed a novel bi-directional promoter (FsFfCBD) by placing two heterogeneous core-promoters from the Figwort mosaic virus sub-genomic transcript promoter (FsCP, -69 to +31) and Cauliflower mosaic virus 35S promoter (CCP, -89 to +1) respectively on upstream (5') and downstream (3') ends of a tri-hybrid enhancer (FsEFfECE), in reverse orientation. The FsEFfECE domain encompasses three heterologous enhancer fragments from Figwort mosaic virus sub-genomic transcript promoter (FsE, 101 bp, -70 to -170), Figwort mosaic virus full-length transcript promoter (FfE, 196 bp, -249 to -54) and Cauliflower mosaic virus 35S promoter (CE, 254 bp, -343 to -90). The bi-directional nature of the FsFfCBD promoter (coupled to GFP and GUS) was established both in transient systems (onion epidermal cells and tobacco protoplasts) and transgenic plant (Nicotiana tabacum samsun NN) by monitoring the simultaneous expression of GFP and GUS employing fluorescence (for GFP) and biochemical (for GUS) based assays. In transgenic plants, the FsFfCBD promoter was found to be 6.8 and 2.5 times stronger than two parent promoters; Fs and FfC respectively. The bi-directional compound promoter FsFfCBD, composed of three heterologous enhancers with enhanced activity could become a valuable additional tool for efficient plant metabolic engineering and molecular pharming.
Asunto(s)
Caulimovirus/genética , Elementos de Facilitación Genéticos , Vectores Genéticos/genética , Regiones Promotoras Genéticas , Biotecnología/métodos , Caulimovirus/metabolismo , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Cebollas/genética , Cebollas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Protoplastos/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
We have isolated a teratocyte secretory protein (TSP14) gene product from a hymenopteran endoparasite that disrupts the growth of lepidopteran insect larvae. To evaluate the insecticidal activity of TSP14 for the protection of crops from insect damage, chimeric gene constructs of TSP14 were expressed in transgenic plants. The coding sequence of the TSP14 gene, with and without its native signal peptide, was placed between the modified peanut chlorotic streak virus (PClSV) full-length transcript (FLt) promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. These chimeric genes, expressed in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) were stably inherited in successive plant generations (R0, R1 and R2 progeny) as shown by molecular analysis. A Western blot analysis of plant extracts showed the presence of a polypeptide of the expected size that cross-reacted with TSP14-specific antibodies. Larvae of the tobacco budworm (Heliothis virescens) and tobacco hornworm (Manduca sexta) which were fed with several independent homozygous transgenic plant lines (R2 progeny) exhibited mortality and reduced growth rates compared to those fed with plants transformed by a vector control. Our results demonstrate the potential for introduction of the TSP14 gene into plants in order to achieve protection against lepidopteran pests.