Biotechnological Applications of Plastid Foreign Gene Expression

Chloroplast is responsible for the major metabolic process photosynthesis. These organelles have their own genome and in the last three decades, the chloroplast genome has been broadly studied and manipulated through genetic engineering tools. The transfer of genes into chloroplast provides advantage over the insertion of transgene into nuclear genome, including overexpression of foreign protein, no positional effects, absence of epigenetic effects and uniparental inheritance of the transgenes, the ability to express multiple transgenes in operons and the possibility of eliminate the marker gene after the transformation and integration of the foreign gene. Now more than 100 transgenes have been reported stably integrated into the chloroplast genome including genes encoding enzymes with industrial value, biomaterials, biopharmaceuticals, vaccines and genes with agronomic trails. The chloroplast genetic tools have been implemented in several important crops. So, the chloroplast engineering technology has been positioned as the most important for the production of proteins and metabolites with biotechnological applications

High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly

Análisis morfológico de la diversidad del pasto navajita [Bouteloua gracilis (Willd. ex kunth) lag. ex steud.], en Chihuahua, México

An efficient morphologic characterization of plants includes evaluation of forage traits. The objective was to analyze morphologic diversity in native populations of blue grama grass [Bouteloua gracilis (Willd. ex Kunth) Lag. ex Steud.] in Chihuahua. In 2006, 173 ecotypes were collected in Chihuahua and transplanted in La Campana Experimental Site. Ecotypes were evaluated through morphological traits and their population structure was defined. Principal component and cluster analysis were used for data analysis. The three first principal components explained 57.3 % of total variation. The PC1, PC2 and PC3 explained 35.3 %, 12.4 % and 9.52 % of the global variation, respectively. Most important variables for PC1 were forage yield, stem density, and plant height, for PC2 were spikelet width, inflorescence length and spikelet number, and for PC3, stem diameter, leaf width and spikelet length. The PC1 included variables related to growth capacity. The PC2 grouped variables associated to propagation and dispersion. The PC3 included variables related to biomass accumulation. Also, four groups were obtained from the 145 established ecotypes. Groups 1, II, III, and IV included 59, 24, 19, and 43 ecotypes, respectively. A high morphological diversity was observed and ecotypes with high forage potential were detected according to their morphological trails. Genetic variability in blue grama grass from Chihuahua is readily available and specimens having excellent forage characteristics could be used in range improvements programs.Una eficiente caracterización morfológica de plantas es el empleo de descriptores forrajeros seleccionados. El objetivo fue analizar la diversidad morfológica de poblaciones nativas del pasto navajita [Bouteloua gracilis (Willd. ex Kunth) Lag. ex Steud.]. En el año 2006 se recolectaron 173 ecotipos de navajita en Chihuahua, y se trasplantaron en el Sitio Experimental La Campana. Los ecotipos establecidos se calificaron mediante descriptores morfológicos y se definió su estructura poblacional. Los datos se analizaron por medio de componentes principales (CP) y conglomerados jerárquicos. Los tres primeros componentes explicaron 57.3 % de la variación total obtenida. El CP1 explicó 35.3 %, el CP2 12.4 % y el CP3 9.52 % de la variación global. Para el CP1 las variables más importantes fueron rendimiento de forraje, densidad de tallos y altura de forraje, para el CP2, grosor de espigas, longitud de inflorescencia y número de espigas y en el CP3, diámetro de tallo, ancho de lámina de hoja y longitud de espiga. El CP1 agrupó variables relacionadas con capacidad de crecimiento. El CP2 incluyó variables que reflejan rasgos de dispersión y propagación. El CP3 reunió variables relacionadas con la asignación de biomasa. Además, se obtuvieron cuatro grupos que integraron a los 145 ecotipos establecidos. El grupo I, II, III y IV integraron 59, 24, 19 y 43 ecotipos, respectivamente. Se presentó alta variabilidad morfológica y se detectaron ecotipos con alto potencial forrajero de acuerdo a su diversidad morfológica. Se dispone de variabilidad morfológica de pasto navajita con atributos forrajeros sobresalientes para ser incluidos en programas de mejoramiento de pastizales

Chloroplasts: The Future of Large-Scale Protein Production

Chloroplast engineering has matured considerably in recent years. It is emerging as a promising tool to address the challenges related to food security, drug production, and sustainable energy posed by an ever-growing world population. Chloroplasts have proven their potential by efficiently expressing transgenes, encapsulating recombinant proteins, and protecting them from cellular machinery, making it possible to obtain highly functional proteins. This quality has also been exploited by interfering RNA technology. In addition to the practical attributes offered by chloroplast transformation, such as the elimination of position effects, polycistronic expression, and massive protein production, the technique represents an advance in biosafety terms; however, even if its great biotechnological potential, crops that have efficiently transformed are still a proof of concept. Despite efforts, other essential crops have remained recalcitrant to chloroplast transformation, which has limited their expansion. In this chapter, we address the most recent advances in this area and the challenges that must be solved to extend the transformation to other crops and become the de facto tool in plant biotechnology

Lactoferrin: A Glycoprotein Involved in Immunomodulation, Anticancer, and Antimicrobial Processes

Lactoferrin is an iron binding glycoprotein with multiple roles in the body. Its participation in apoptotic processes in cancer cells, its ability to modulate various reactions of the immune system, and its activity against a broad spectrum of pathogenic microorganisms, including respiratory viruses, have made it a protein of broad interest in pharmaceutical and food research and industry. In this review, we have focused on describing the most important functions of lactoferrin and the possible mechanisms of action that lead to its function

Biocombustibles: estrategias limpias para combatir la crisis energética

México se encuentra ante una eventual crisis energética debido a la reducción en sus reservas probadas del petróleo, lo que ha tenido como consecuencia un incremento en los precios de los combustibles. Además, la utilización de este hidrocarburo ha generado emisión de gases con efecto invernadero, contribuyendo al cambio climático. Para solucionar este problema se requiere desarrollar tecnologías alternativas que nos permitan sustituir los combustibles derivados del petróleo. Estos hechos hacen evidente la necesidad de utilizar fuentes alternas de energía. Los biocombustibles son recursos energéticos producidos por el ser humano a partir de materias generadas por seres vivos, a las cuales se les denomina “biomasa”. Esta segunda generación de biocombustibles plantea el uso de lignocelulosa, que es el polímero más abundante sobre la superficie del planeta; para lograrlo se requiere del desarrollo biotecnológico que permita la despolimerización efectiva de la biomasa vegetal. Abstract Mexico is facing a possible energy crisis due to the reduction in proven reserves of oil and consequently, it has resulted in the increase in fuel prices. To solve this problem it requires developing alternative technologies that allow us to replace fuels from petroleum. Besides that, the use of this fuel has generated greenhouse gases emissions, contributing to the climate change. These facts make clear the need for alternative energy sources. Biofuels are a type of fuel whose energy is derived from living organisms called “biomass” and produced by human beings. These second-generation biofuels require the use of lignocellulose that is the most abundant polymer on the surface of the planet, to get this, it is required the development of a biotechnology process that allows the effective depolymerization of plant biomass

Isolation of microRNAs from Bouteloua gracilis chlorophyllous cells and their in silico characterization

Bouteloua gracilis es un pasto nativo del norte de México, que es utilizado como fuente de forraje para el ganado por su alto contenido nutritivo; tiene elevada tolerancia al estrés osmótico que le permite vivir en climas secos; sin embargo, los mecanismos moleculares que le confieren esta tolerancia aún no se han reportado. Existe una clase de RNAs pequeños (sRNAs) llamados microRNAs (miRNAs), que se unen por complementariedad a RNAs mensajeros blanco, etiquetándolos para su degradación o supresión de la traducción. En este trabajo se reporta el aislamiento de sRNAs de B. gracilis, a través de la clonación de concatámeros y su secuenciación. El análisis in silico de la secuencias, permitió la identificación de miRNAs conservados en B. gracilis y reportados en Populus trichocarpa, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, Zea mays, Malus domestica y Linum usitatissimum.Además, se predijo la estructura secundaria de los precursores de dos miRNAs (pre-miRNAs), usando como referencia los genomas de Glycine max, Zea mays, Sorghum bicolor y Oryza sativa. Finalmente, se identificaron seis mRNAs blanco para uno de ellos. La identificación de miRNAs en Bouteloua gracilis, ayudará a comprender como estas moléculas regulan la expresión genética en esta especie, y sus relaciones con los mecanismos de respuesta a estrés ambiental.Bouteloua gracilis is a grass native to Mexico, it is used as forage source for livestock because of its high nutritional value. It has high tolerance to osmotic stress and therefore can live in arid zones; however, regulation mechanisms in gene expression that confer these characteristics have not been reported. There is a class of small RNAs (sRNAs) called microRNAs (miRNAs), which regulate gene expression. They are complementary and act by binding to Messenger RNAs (mRNAs to inhibit translation or by degrading them. In this work, the isolation of sRNAs from B. gracilis through cloning and sequencing of concatemers is reported. In silico analysis of the sequences obtained allowed to identify conserved sequences in Populus trichocarpa, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, Zea mays, Malus domestica, and Linum usitatissimum. Furthermore, the secondary structure of the miRNA precursor (premiRNA) was predicted from two sequences isolated from In silico analysis using Glycine max, Zea mays, Sorghum bicolor, and Oryza sativa as reference genomes. Finally, six target mRNAs were identified for one of the miRNAs obtained. Identification of miRNAs in Bouteloua gracilis will help to understand how these molecules regulate gene expression and in the future will allow for the study at molecular level, providing insight on how this grass responds to environmental stress

Manejo biotecnológico de gramíneas forrajeras

Biotechnological advances achieved in basic crops and model plants are currently being applied to practically all grasses, being them cereals, forages, turfs, ornamentals or species having industrial or pharmaceutical importance. An unprecedented revolution in possibilities for manipulating graminaceous species through current and coming technologies can be expected which should impact all areas and levels of knowledge and use of these species. In the present study, the importance, biology, perspectives and scientific advances reached in forage grasses within global biotechnology of Poaceae are described. The possibility for analyzing and altering the genome through an ever increasing number of techniques for scrutiny and delivering DNA is emphasized, because of its potential contribution to help explain some of the ecological and physiological enigmas in these plants not yet cleared up, as well as to foster an increase in productivity and an improvement of their agronomic characteristics. Finally, some of the biotechnological advances accomplished in one of the most important forage grasses of Mexico, blue grama (Bouteloua gracilis), are described. Biotechnology is expected to improve our knowledge on the blue grama genetic variability base in native populations and to allow for the development of strategies aimed at the transfer into cereals of some of the genes that confer high tolerance to water stress in B. gracilis.Los avances biotecnológicos alcanzados en cultivos agrícolas y plantas modelo están siendo actualmente extendidos a prácticamente todas las gramíneas, sean éstas cereales, zacates forrajeros, pastos de césped, plantas ornamentales o especies con importancia en el sector industrial o farmacéutico. Se vislumbra una revolución sin precedente en las posibilidades para la manipulación de las gramíneas mediante las tecnologías actuales o emergentes, que impactarán todas las áreas y niveles del conocimiento y uso de estas especies. En esta contribución se establece la importancia, biología, perspectivas de estudio y los avances alcanzados en los zacates forrajeros dentro de la biotecnología global de las Poaceae. La posibilidad de analizar y alterar el genoma de las plantas a través del creciente número de técnicas de escrutinio y transferencia de ADN recibe especial atención, en vista de las contribuciones potenciales que pueden tener a la explicación de muchos de los enigmas hasta hoy no esclarecidos en la ecología y fisiología de estas plantas, así como al incremento de la productividad y manipulación de sus características agronómicas. En un último apartado, se describen algunos de los avances biotecnológicos logrados hasta el momento en una de las gramíneas forrajeras más importantes de México, navajita azul (Bouteloua gracilis). Se espera que los estudios biotecnológicos mejoren nuestro conocimiento sobre las bases de la variabilidad genética de las poblaciones nativas de navajita azul, y permitan el diseño de estrategias enfocadas a la transferencia de algunos de los genes que confieren a B. gracilis su elevada tolerancia al estrés hídrico, a especies pertenecientes al grupo de los cereales