SINOPSIS
Justificación
En México y Sinaloa se posiciona como un cultivo de gran importancia para el consumo nacional e internacional (Faostat, 2015a, Faostat, 2015b, Siap-Sagarpa, 2012). La producción de este cultivo puede verse influenciada por condiciones bióticas y abióticas, que provocan una interacción funcional con el metabolismo de la planta, ya sea primario o secundario. La adaptación de las plantas a la deficiencia de cada elemento es variada, pero una de las estrategias coincidentes es el aumento de la cantidad de transportadores en la membrana para aumentar la capacidad de asimilación de los elementos requeridos y mantener la homeostasis celular, por lo que se debe realizar el estudio de estos mecanismos. es importante para un mejor conocimiento básico de la nutrición del tomate.
El uso continuo de fertilizantes promueve fenómenos desfavorables para el suelo, como la pérdida de microorganismos de importancia agrícola, contaminación y polución de las reservas de aguas subterráneas, además de los costos asociados a la adquisición de estos insumos agrícolas. En esta última área del conocimiento, es importante comprender los mecanismos por los cuales las plantas responden a un estrés determinado para poder crear estrategias hacia una agricultura inteligente y eficiente. En este sentido, existen vacíos en el conocimiento sobre los mecanismos implicados en la respuesta a la deficiencia de Zn y P en tomate.
Por ello, consideramos de gran importancia ampliar el conocimiento actual sobre estas y otras familias de proteínas.
Antecedentes
- Descripción Genética del Tomate
- Tomate Como Organismo Experimental
- Deficiencias Nutrimentales
- Deficiencias De Fósforo en Plantas
- Deficiencia de Zinc en Plantas
- Estrategias para la identificación y caracterización de proteínas
- Bases de datos
- Alineamientos múltiples
- Dominios transmembranales, dominios conservados y péptido señal
- Proteínas PHT1 y ZIP en otros modelos de estudio
Varios de los genes de la familia ZIP codifican transportadores de Zn (Grotz et al., 1998). Comprender estas diferencias entre los distintos tomates analizados es fundamental para acelerar la mejora tradicional o genética (Lin et al., 2014). En Brassica nigra, ante una deficiencia de P, disminuyen los niveles de Pi, ATP, ADP, Fru-2,6-P2 (Fructosa 2,6-bisfosfato) y PPi, mientras que aumenta la cantidad de aminoácidos libres; Además, la actividad de las enzimas PFP (PPi: D-fructosa 6-fosfato 1-fosfotransferasa), NAPD-G3PDH no fosforilante, PEP (fosfoenolpiruvato) fosfatasa y PEP (fosfoenolpiruvato) carboxilasa aumenta alternativamente para generar glucosa. (Duff et al., 1989).
Dentro de los genes de respuesta diferencial fisiológica se destacaron los transportadores y los factores de transcripción (Rubio et al., 2001; Nilsson et al., 2007), elementos importantes y relevantes para la. En caso de deficiencia de fósforo, el factor de transcripción PHR1 se activa, permitiendo la regulación de los genes PSI (Phosphate Starvation Induced), incluidos los genes de la familia Pht1 (Bari et al., 2006). La distribución tisular del gen Pht1;4 es múltiple y su expresión depende de los órganos con disponibilidad de P (Misson et al., 2004).
La regulación de esta familia se produce mediante la activación del complejo dimérico b19/b23 en respuesta a niveles bajos de Zn; Una vez activo, el complejo actúa como factor de transcripción (Assunção et al., 2014).
Hipótesis
Algunos de los genes expresados en la deficiencia de fósforo y zinc ya se han caracterizado en diferentes especies, y se ha demostrado que la similitud de estas secuencias con Arabidopsis es un indicador de homología en su función. La base de datos NCBI muestra que secuencias con un porcentaje de similitud de hasta el 50% presentan productos con funciones similares, incluso en especies de diferentes familias. Las preguntas de investigación son: ¿Cuáles son las características (tamaño, identidad, dominios conservados) de las secuencias SlZIP2, SlZIP3, SlZIP4, SlZIP5, SlZIP5 X1, SlZIP7, SlZIP8?
¿Cuáles son las características (tamaño, identidad, dominios conservados) de las secuencias Pht1;11 X1, Pht1;11 X2? ¿Cuáles serán los niveles de expresión relativos de genes seleccionados de tomate de la familia ZIP en condiciones de nutrición diferencial con zinc? 26 peso molecular entre 36 y 40 kDa y un punto isoeléctrico entre 6 y 8 3) Las secuencias candidatas a ser transportadoras de Zn en tomate presentarán niveles de expresión relativos más altos en condiciones de deficiencia de zinc.
Objetivo General
Objetivos Específicos
Sección Integradora
Mudge S, Rae A, Diatloff E, Smith W (2002 ) Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis. These results suggest a coordinated system of ZIP proteins in tomato depending on the time of exposure to (-Zn) stress. In eukaryotes, it is estimated that about 9% of the proteome is Zn-dependent in various ways, with most of these proteins having enzymatic or transcriptional functions (Andreini et al., 2009).
Nucleotide sequences of confirmed members of the ZIP family of proteins from different plants (Arabidopsis thaliana, Populus trichocarpa, Medicago truncatula) were introduced into the NCBI (National Center for Biotechnology Information) Blast (Basic Local Alignment Search Tool) algorithm to identify similar sequences in tomato (S. lycopersicum L.). 62 the presence of the putative domain Zip superfamily (cl00437) present in zinc transporter members of the ZIP family. The signal peptides of the proteins encoded by the evaluated genes were identified using targetP 1.1 to determine their localization: chloroplastic, mitochondrial or secretory pathway (Emanuelsson et al., 2007).
The EF1α gene was used as a control and ZIP gene expression values were calculated relative to control plants (0 h) using the method of Pfaffl (2001). Clustering of proteins in both monocots and clades may be an indication of their conserved functions (Tiong et al., 2015). SlZIP4 was predicted to be a chloroplastic protein and did not form a defined clade; however, it shares characteristics with other members of the ZIP protein family.
Some members of the ZIP protein family are overexpressed by Zn deficiency in response to unfavorable conditions (Grotz et al., 1998; Jain et al., 2013). Several studies have shown an increase in transcript levels of genes encoding mineral transporters under mineral deficiency, including some members of the ZIP proteins in several species. In this work, two members of the ZIP family (ZIP5 and ZIP8) were overexpressed under Zn deficiency (0 µM) in tomato (Solanum lycopersicum cv. Micro-Tom) leaves, and the fold change was correlated with the time the plants were exposed lack.
Elevated expression of metal transporter genes in three accessions of the metal hyperaccumulator Thlaspi caerulescens. Identification and characterization of zinc-regulated transporters, the iron-regulated transporter-like protein (ZIP) gene family in maize. Identification and characterization of several new members of the ZIP family of metal ion transporters in Medicago Truncatula.
Cloning and functional identification of two members of the ZIP (Zrt, Irt-like protein) gene family in rice (Oryza sativa L.). Unrooted analyzes of homologous proteins of the ZIP family with MEGA 6 with neighbor-joining with bootstrap values. Rae AL.; Cybinski DH.; Jarmey JM y Smith FW (2003) Characterization of two phosphate transporters from barley.; evidence for diverse function and kinetic properties among members of the Pht1 family.
THE ROLE OF ZIP PROTEINS ON ZINC ASSIMILATION AND
PHOSPHORUS TRANSPORTER PROTEINS FROM THE PHT1 FAMILY
In silico analyzes showed the presence of the conserved Zip superfamily domain (cl00437), 6 to 9 putative transmembrane (TM) domains, a length between 355–490 amino acids, and a histidine-rich “variable region”. The relative expression of the ZIP4 gene remained constant under Zn deficiency during all times evaluated, while ZIP5 showed an expression peak after 6 h and then decreased after 48 h. Optimal growth of this crop requires an adequate mineral supply and zinc (Zn) is one of the most important micronutrients as it is essential in processes such as CO2 fixation, photosynthesis (Sasaki et al., 1998) and protein biosynthesis (Prask and Plocke, 1971).
Some genes encoding members of the ZIP family of proteins (required for zinc influx) have been characterized in several species; they have shown upregulation at the transcriptional level under Zn deficiency, some with ~200-fold change, to make the transport of this element into the cytoplasm from outside the cell more efficient ( Grotz et al., 1998 ; Jain et al. , 2013). To determine the evolutionary relationship between the ZIP proteins, a phylogenetic tree was constructed using MEGA 7.0 with the Neighbor Joining algorithm and a bootstrap test with 1000 replicates. The model used was the crystal structure of Bordetella bronchiseptica with bound Zn2+ Zrt-/lrt-like protein (PDB 5TSA) and the figure was built using PyMol (Schrodinger, 2019).
The characteristics of the putative ZIP family proteins, such as predicted transmembrane domains, molecular weight, isoelectric point and signal peptide, are shown in Table 2. The length of the five proteins ranged between 337 and 407 amino acids with isoelectric points between 5.8 and 8.4, which are similar to those reported for ZIP proteins previously characterized in silico (Guerinot, 2000; Pedas et al., 2009). However, it is worth noting that the temporal and tissue-specific expression of genes encoding these proteins is associated with regulatory mechanisms involving non-coding sequences.
Phylogenetic analyzes of candidate ZIP proteins from tomato indicated the presence of clades with amino acid sequences from another species based on sequence homology; for example, SlZIP2 was grouped with MtZIP2 while SlZIP3 was in a clade with MtZIP1 and AtZIP1. These patterns appear to depend on the time elapsed since the onset of stress, but may also be. 68 related to the level of mineral nutrition, meaning they can be activated by a threshold, all as part of the plant's strategy to maintain homeostasis.
The models were built using the crystal structure of Bordetella bronchiseptica with bound Zn2+ Zrt-/lrt-like protein (PDB 5TSA) and the figure was built using PyMol. Phosphorus is a fundamental element for the optimal development of plants, as it is involved in structural and energetic processes. One of the plant's most important strategies to maintain homeostasis under P deficiency is the production of membrane transporters of the PHT1 family of proteins, eight members of which have been characterized in tomato (Solanum lycopersicum L.). However, two sequences without previous characterization were found in the database.
The main objective of this work was to identify and characterize two isoforms of PHT1 family proteins in tomato. Wu Z.; Zhao J.; Gao R.; Hu G.; Gai J.; Xu G y Xing H (2011) Molecular cloning, characterization and expression analysis of two members of the Pht1 phosphate transporter family in glycine max. Jan W.; Chen G-H.; Yang L-F.; Gai J-Y y Zhu Y-L (2014) Overexpression of the rice phosphate transporter gene OsPT6 increases tolerance to low phosphorus stress in plant soybean.
EXPRESSION PATTERNS OF ZIP PROTEINS UNDER ZINC DEFICIENCY
IDENTIFICACIÓN Y CARACTERIZACIÓN IN SILICO DE DOS