Método para la extracción de ADN cloroplastídico de Bouteloua gracilis como herramienta para aplicaciones moleculares
Method of chloroplast DNA extraction from Bouteloua gracilis as a tool for molecular applications
Resumen
La manipulación genética del genoma de cloroplasto esta a la vanguardia en investigación biotecnológica. El pasto B. gracilis es un modelo para el estudio de estrés hídrico y oxidativo, capacidades relacionadas al cloroplasto. Es necesario tener un método que facilite la obtención de ADN de buena calidad, particularmente cuando se refiere al ADN de cloroplasto de B. gracilis. La implementación de un método para la extracción de cpADN se logró probando diferentes estrategias de extracción de cpADN reportados en la literatura y combinando las etapas más apropiadas para B. gracilis. La incorporación de un paso adicional del lavado con CTAB permitió la recuperación de cpDNA enriquecido, el cual se puede utilizar en el desarrollo de hermanitas moleculares. Debido la implementación de un protocolo de extracción también fue posible obtener una cantidad considerable de cpADN de B. gracilis. El cpADN fue digerido con varias enzimas de restricción y los fragmentos resultantes fueron analizados por Southern blot con las sondas de los genes de rADN 16S y 23S. Abstract Genetic manipulation of the chloroplast genome is at the forefront of biotechnology research. B. gracilis is a model for the study of water stress and oxidative capacities related to the chloroplast. It is requires a method to facilitate the obtaining of good quality DNA, particularly when it comes to chloroplast DNA of B. gracilis. The implementation of a method for extracting cpDNA was achieved by assaying different strategies cpDNA extraction reported in the literature and combining the most appropriate stage for B. gracilis for cpDNA extraction. The incorporation of an additional step of washing with CTAB after lysis of chloroplasts, allowed the recovery of enriched cpDNA which can be used in the development of molecular tools. Due to the implementation of an extraction protocol was also possible to obtain a considerable amount of B. gracilis cpDNA. The cpDNA was digested with several restriction enzymes and the resulting fragments were analyzed by Southern blot with probes of genes rADN 16S and 23S.Citas
Aguado-Santacruz, G.A., D.A. Betancourt-Guerra, T. Siquerios- Cendón, S.Arévalo-Gallegos, B. Rivera-Chavira, G.V. Nevárez- Moorillon, B.M. Moreno-Gómez & Q. Rascón-Cruz. 2011. Comparison of the structure and organization of the rrna operons of Bouteloua gracilis and Zea mays. Canadial Journal of Plant Science 91(1):107-116. https://doi.org/10.4141/cjps10089
Blaustein, D.R., Johnson & D. Mathieu. 1998. Biology. The dynamics of life. Glencoe/McGraw-Hill. ISBN 0028254317, 9780028254319.
Bogorad, L. 2000. Engineering chloroplasts; an alternative site for foreign genes, proteins, reactions and products. Trends in Biotechnology 18(6):257-263. https://doi.org/10.1016/S0167-7799(00)01444-X
Bookjans, G., B.M. Stummann & K. Henningsen. 1984. Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic-strength. Analytical Biochemistry 141(1): 244-247.
Chebolu, S. & H. Daniell. 2007. Stable expression of GAL/GALNAc lectin of Entamoeba histolytica in transgenic chloroplast and immunogenicity in mice towards vaccine development for amebiasis. Plant Biotechnology Journal 5(2):230-239. https://doi.org/10.1111/j.1467-7652.2006.00234.x
Daniell, H., M.S. Khan & L. Allison. 2002. Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends in Plant Science 7(2):84-91. http://dx.doi.org/10.1016/S1360-1385(01)02193-8
Diekmann, K., T. Hodkinson, E. Fricke & S. Barth. 2008. An optimized chloroplast DNA extraction protocol for grasses (Poaceae) proves suitable for whole plastid genome sequencing and SNP detection. PLoS ONE 3(7):e2813. https://doi.org/10.1371/journal.pone.0002813
García-Sánchez, R. & A. Monroy-Ata. 2005. Micrositios del pasto navajita (Bouteloua gracilis) en comunidades de pastizal y de matorral del altiplano mexicano. Revista Especializada en Ciencias Químico-Biológicas 8(2): 61-70. https://www.redalyc.org/articulo.oa?id=43220801
Hoisington, D., M. Khairallah & D. González-De León. 1994. Laboratory protocols: CIMMYT Applied Molecular Genetics Laboratory. Centro Internacional de Mejoramiento de Maíz y Trigo. https://repository.cimmyt.org/handle/10883/1333
Jansen, R.K., L.A. Raubeson, J.L. Boore, C.W. de Pamphilis, T.W. Chumley, R.C. Haberle, S. K. Wyman,A. J.Alverson, R. Peery, S. J. Herman, H. M. Fourcade, J. V. Kuehl, J. R. McNeal, J. Leebens-Mack & L. Cui. 2005. Methods for obtaining and analyzing whole chloroplast genome sequences. Methods in Enzymology 395(2005): 348-384. https://doi.org/10.1016/S0076-6879(05)95020-9
Kessler, C., H. Höltke, R. Seibl, J. Buró & K. Mühlegger. 1990. Non-radioactive labeling and detection of nucleic acids. A novel DNA labeling and detection system based on digoxigenin: Anti-digoxigenin ELISA Principle (digoxigenin system). Biological Chemistry Hoppe-Seyler 371(10):917-927. https://doi.org/10.1515/bchm3.1990.371.2.917
Kolodner, R. & K.K. Tewari. 1979. Inverted repeats in chloroplast DNA from higher plants. Proceedings of the National Academy of Sciences 76(1): 41-45. https://doi.org/10.1073/pnas.76.1.41
Koya, V. & H. Daniell. 2005. OBPC symposium: maize 2004 & beyond – Recent advances in chloroplast genetic engineering. In vitro Cellular & Developmental Biology – Plant 41(4):388-404. https://doi.org/10.1079/IVP2005660
Leister, D. 2003. Chloroplast research in the genomic age. Trends in Genetic 19(1): 47-56. https://doi.org/10.1016/s0168-9525(02)00003-3
Palmer, J.D. 1987. Chloroplast DNA evolution and biosystematic uses of chloroplast DNA variation. The American Naturalist 130(Suppl. Plant Molecular Evolution):S6-S29. https://www.journals.uchicago.edu/doi/abs/10.1086/284689?journalCode=an
Ruíz, G. 2002. Optimization of codon composition and regulatory elements for expression of the human IGF-1 in transgenic chloroplasts (Thesis, University of Central Florida).
Sager, R. & M. R. Ishida. 1963. Chloroplast DNA in Chlamydomonas. Biochemistry 50(4): 725-730. https://doi.org/10.1073%2Fpnas.50.4.725
Scott, S.E. & M. J. Wilkinson. 1999. Low probability of chloroplast movement from oilseed rape (Brassica napus) into wild Brassica rapa. Natural Biotechnology 17(4):390-392. http://dx.doi.org/10.1038/7952
Southern, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98(3):503-517. https://doi.org/10.1016/S0022-2836(75)80083-0
Sugiura, M. 1992. The chloroplast genome. Plant Molecular Biology 19(1):149- 168. https://doi.org/10.1007/bf00015612
Tregoning, J.S., P. Nixon, H. Kuroda, Z. Svab, S. Clare, F. Bowe, N. Fairweather, J. Ytterberg, K. Van Wijk, G. Dougan & P. Maliga. 2003. Expression of tetanus toxin fragment C in Tobacco chloroplasts. Nucleic Acid Research 31(4):1174-1179. https://doi.org/10.1093/nar/gkg221
Triboush, S.O., N.G. Danielenko & O.G. Davydenko. 1998. A method for isolation of chloroplast DNA and mitochondrial DNA from sunflower. Plant Molecular Biology 16(2):183-189. https://doi.org/10.1023/A:1007487806583
Virupakshi, S. & G. Naik. 2007. Purification of DNA from chloroplast and mitochondria of sugarcane. Current Science 92(11):1613-1619. https://www.currentscience.ac.in/Volumes/92/11/1613.pdf
Derechos de autor 2020 TECNOCIENCIA Chihuahua
Esta obra está bajo licencia internacional Creative Commons Reconocimiento-NoComercial 4.0.
