Stable transformation of Lettuce : towards therapeutic protein production
| Autor: | Silvestre, Joana Margarida Martins |
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| Přispěvatelé: | Gregory, Franklin, Dias, Alberto Carlos Pires, Universidade do Minho |
| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: | |
| Zdroj: | Repositório Científico de Acesso Aberto de Portugal Repositório Científico de Acesso Aberto de Portugal (RCAAP) instacron:RCAAP |
| Popis: | Dissertação de mestrado em Biotecnologia e Bioempreendedorismo em Plantas Aromáticas e Medicinais Use of plants as bioreactors for protein production was being increasing in the recent years, although the methods presently used are not efficient, easy and cost effective. The purification of the proteins produced using plant bioreactors still remains a challenge and very expensive. Elastin-like polypeptides (ELPs), which exhibit a potentially useful property of a thermally responsive reversible phase transition, can help in protein purification, one of the most expensive steps of heterologous protein production in plants. Lactuca sativa, belongs to Asteraceae family, is one of the most used salad vegetable. In order to express any gene of our interest, it is essential to have an efficient method of DNA delivery coupled with an efficient regeneration protocol. Hence, with the major goal of expressing genes encoding therapeutic proteins fused with a reporter gene in lettuce, we have established an efficient protocol for lettuce regeneration and transformation. Since the efficient recovery of transgenic plants is based on the robustness of the regeneration system, we first studied different parameters such as age, type of explant, genotype and plant growth regulator (PGR) regimen to improve plant regeneration. Briefly, various explants were cultured on MS medium supplemented with different PGR combinations. Our results clearly show that combination of 0.1mg/L BA and 0.1 mg/L NAA could induce efficient shoot regeneration. Among the different varieties tested, SBR showed the highest induction of shoots with statistical significance. When different types of explants from the four varieties were cultured on MS medium containing 0.1mg/L BA and 0.1mg/L NAA, efficient shoot regeneration was observed from cotyledon explants. The age of explants also affected regeneration efficiency, which is demonstrated by the difference between explants obtained from 7 and 28 days old seedlings in their regeneration potential. To standardize a robust protocol for improved A. tumefaciens mediated transformation of L. sativa, we analyzed different parameters such as seed germination on medium containing different concentration of 2,4-D, ultrasonication (US) of explants for various durations and supplementation of different concentration of acetosyringone (AS) in the co-cultivation medium. Seed germination on medium supplemented with 1.0 mg/L 2,4-D increased the infection and the number of shoots per cotyledon explant. US for 2 minutes increased the infection percentage. However, the presence of of acetosyringone (AS) in the co-cultivation medium did not show a significant impact on A. tumefaciens infection and regeneration. Optimization of all the above parameters allowed us to successfully transform lettuce varieties by A. tumefaciens harboring plant expression vector pC2301. As the result, three transgenic plants were obtained. These transgenic plants grew normally and eventually produced viable seeds after flowering. Histochemical GUS assay and molecular analysis of the next generation (T1) seedlings revealed the presence of transgenes indicating that the transgenes were stably integrated into the lettuce genome and segregated according to the Mendelian pattern (3:1). Using the standardized transformation protocol, we were able to introduce gene encoding GFP-ELP fusion protein into lettuce genome after transformation with A. tumefaciens harboring the binary vector pGE. However, characterization of transgenic plants and purification of GFP protein using the temperature transition property of ELP are currently underway. In conclusion, the present study demonstrates the feasibility of expressing reporter GFP gene linked to ELP in lettuce, which opens the door for efficient production of therapeutic proteins as fusions with ELP in lettuce. O uso de plantas como biorreactores na produção de proteinas tem vindo a aumentar. Contudo, os métodos usados apresentam ainda várias limitações, como a falta de eficiencia e um custo elevado. Desta forma a produção de proteinas utilizando plantas como biorreacetores permanence um desafio. Polipeptidos tipo elastina (ELP do inglês “Elastin-lyke polypeptides”) possuem a vantagem de resposta à temperatura usando uma fase de transição reversível. Esta característica permite uma eficiente purificação proteica, considerado ainda um dos passos mais caros na produção de proteínas em plantas. Desta forma torna-se crucial desenvolver um protocolo eficiente para a regeneração e transformação de plantas com vista ao aumento da produção e pureza proteica. Lactuca sativa (alface), pertencente à família Asteraceae é um dos vegetais mais consumido mundialmente. De forma a desenvolver um protocolo eficaz para a regeneração de alface, foram estudados vários parâmetros como a idade e tipo de explante, genótipo e reguladores de crescimento. Os explantes foram cultivados em meio MS suplementado com diferentes combinações de reguladores de crescimento. De todas, a combinação de 0,1mg/L BA + 0,1mg/L NAA induziu, com significância estatística, maior produção de rebentos nos cotilédones da variedade SBR. A regeneração também foi afectada pela idade dos explantes. De uma forma geral verificou-se uma diminuição na capacidade de regeneração dos explantes mais novos (7 dias) para os mais velhos (28 dias). Esta diferença mais notória para as variedades SBR e BL. De forma a standardizar um protocolo para melhorar a infecção de alface por A. tumefaciens, foram analizados diferentes parâmetros químicos (2,4-D e AS) e físicos (US). O meio de germinação com 1,0 mg/L de 2,4-D aumentou tanto a infecção como o numero de rebentos por explante (p |
| Databáze: | OpenAIRE |
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