Identificación de promotores exclusivos de polen en arabidopsisthaliana y su posterior aplicación en la obtención de plantas con esterilidad masculina
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Fecha
2013
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
La utilización de cultivos transgénicos en la industria agrícola, desde su liberación comercial
en 1996, se ha incrementado a tal nivel que en algunos casos -como el cultivo de Soya en
Argentina- ha desplazado a los cultivos convencionales. Si bien los cultivos transgénicos poseen
ventajas a nivel de producción, como la resistencia a herbicidas o a herbívoros, uno de los riesgos
asociado a la utilización de cultivos genéticamente modificados es la posibilidad de que ocurra
polinización cruzada con especies silvestres compatibles, adquiriendo estas el transgen. Una de las
estrategias empleadas para evitar la dispersión del polen y posterior polinización es la utilización de
plantas estériles. Esto es posible lograrlo mediante la ablación específica del polen, que consiste en
la eliminación específica de un tejido mediante la expresión de una citotoxina bajo el control de un
promotor finamente regulado. Por esta razón, la identificación de promotores que confieran
expresión exclusiva en polen y su posterior uso para controlar la transcripción de una ribonucleasa
(barnasa) generaría la ablación específica del polen. Mediante la búsqueda en bases de datos de
microarreglos de patrones de acumulación de transcritos que sugirieran expresión exclusiva en
polen, se identificaron cinco genes candidatos, los cuales se denominaron POLLEN SPECIFIC
GENEJ-5 (PSG 1-5). Para respaldar la posibilidad que estos genes estén regulados a nivel
transcripcional se realizó una búsqueda in silico de secuencias sobrerrepresentadas en las regiones
río arriba de los genes PSG; entre los cuales se encontró un elemento de regulación cis descrito en
genes con expresión en polen (LeLAT52) . Los datos entregados por los microarreglos fueron
confirmados mediante RT-PCR en diferentes tejidos (raíz, tallo, hoja, flor cerrada, flor abierta y
polen), y sólo se observó presencia del mRNA en flores y polen. Con la finalidad de determinar la
regulación espacio-temporal que ejercen los promotores de estos genes, se realizó una fusión
transcripcional de las regiones río arriba de los genes PSG con el gen reportero GUS y se
transformaron plantas de Arabidopsis thaliana de manera estable. Al realizar el ensayo GUS fue
posible apreciar actividad GUS exclusivamente en polen para los promotores de los genes PSG2,
PSG4 y PSG5, mientras que PSGJ y PSG2 presentaron actividad GUS en tejidos distintos al
gametofito masculino. Se transformaron plantas de Arabidopsis thaliana de manera estable con un
vector que contiene una fusión transcripcional del los promotores PSG2 y PSG4 a la región
codifican te de la ribonucleasa barnasa y se obtuvieron plantas hemicigotas con un 50% y un 100%
de polen muerto, logrando así la ablación específica del gametofito masculino, lo que demuestra la
gran especificidad de los promotores identificados.
The use of transgenic crops in the agricultura! industry, since the commercial release in 1996, has increased to such levels that in sorne cases -like the soybean in Argentina- it has displaced conventional crops. E ven thought they ha ve advantages in terms of production, such as resistan ce to herbicides or herbivores, one of the risks associated with the use of biotech crops is the possibility of cross-poliination with wild relatives acquiring the transgenes. One of the strategies used to prevent palien spread and pollination is the use of sterile plants, which is possible to achieve by specific ablation of palien, That involves elimination of a specific tissue with the expression of a cytotoxin under the control of a tightly regulated promoter. For this reason, the identification of promoters conferring exclusive expression in palien and subsequent use of these to control the transcription of a ribonuclease (barnase) generate polien-specific ablation. By searching in microarrays databases for transcript accumulation pattern that suggest exclusive expression in palien, five candidate genes were identified, which were designated POLLEN SPECIFIC GENEJ-5 (PSG 1-5). To support the possibility that these genes are regulated at promoter leve!, we performed an in silico search for overrepresented sequences in the upstream regions of the PSG genes, among these was found a cis regulatory element described in genes expressed in palien (LeLAT52). The data provided by the microarrays was confirmed by RT-PCR in diferent tissues (root, stem, leaf, closed flower, open flower and palien), where only the presence of mRNA was observed in flowers and palien. In arder to determine the spatio-temporal regulation exerted by the promoters of these genes, a transcriptional fusion was made of the upstream regions of PSG genes with the GUS reporter gene and transformed stably Arabidopsis thaliana plants. When performing the GUS assay, activity was appreciated exclusively in palien for the promoters of the PSG2, PSGS and PSG4 genes, while PSG2 and PSG 1 showed staining in different tissues. Arabidopsis thaliana plants where stably transformed with a vector containing a transcriptional fusion of the PSG2 and PSG4 promoters with the ribonuclease barnase coding region, obtaining hemicygots plants with 50% and 100% dead palien, achieving mal e gametophyte specific ablation, showing high specificity of the identified promoters.
The use of transgenic crops in the agricultura! industry, since the commercial release in 1996, has increased to such levels that in sorne cases -like the soybean in Argentina- it has displaced conventional crops. E ven thought they ha ve advantages in terms of production, such as resistan ce to herbicides or herbivores, one of the risks associated with the use of biotech crops is the possibility of cross-poliination with wild relatives acquiring the transgenes. One of the strategies used to prevent palien spread and pollination is the use of sterile plants, which is possible to achieve by specific ablation of palien, That involves elimination of a specific tissue with the expression of a cytotoxin under the control of a tightly regulated promoter. For this reason, the identification of promoters conferring exclusive expression in palien and subsequent use of these to control the transcription of a ribonuclease (barnase) generate polien-specific ablation. By searching in microarrays databases for transcript accumulation pattern that suggest exclusive expression in palien, five candidate genes were identified, which were designated POLLEN SPECIFIC GENEJ-5 (PSG 1-5). To support the possibility that these genes are regulated at promoter leve!, we performed an in silico search for overrepresented sequences in the upstream regions of the PSG genes, among these was found a cis regulatory element described in genes expressed in palien (LeLAT52). The data provided by the microarrays was confirmed by RT-PCR in diferent tissues (root, stem, leaf, closed flower, open flower and palien), where only the presence of mRNA was observed in flowers and palien. In arder to determine the spatio-temporal regulation exerted by the promoters of these genes, a transcriptional fusion was made of the upstream regions of PSG genes with the GUS reporter gene and transformed stably Arabidopsis thaliana plants. When performing the GUS assay, activity was appreciated exclusively in palien for the promoters of the PSG2, PSGS and PSG4 genes, while PSG2 and PSG 1 showed staining in different tissues. Arabidopsis thaliana plants where stably transformed with a vector containing a transcriptional fusion of the PSG2 and PSG4 promoters with the ribonuclease barnase coding region, obtaining hemicygots plants with 50% and 100% dead palien, achieving mal e gametophyte specific ablation, showing high specificity of the identified promoters.
Notas
Tesis (Magister en Biotecnología)
Proyecto FONDECYT N° 1120766
Proyecto UNAB DI-74-12/R.
Proyecto FONDECYT N° 1120766
Proyecto UNAB DI-74-12/R.
Palabras clave
Germinación, Ribonucleasa, Arabidopsis Thaliana