- Email: [email protected]
Differential expression of genes possibly involved in incompatible interaction between barley and powdery mildew
Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
that S. coronopifolius and A. armata produce biomolecules with high antitumoral potential. doi:10.1016/j.copbio.2011.05.113
OP43 Obtaining new flower colors by RNA interference technique Sebnem Kavakli Biotechnology Department, Graduate School of Natural and Applied Sciences, Ege University, Izmir, Turkey E-mail address: [email protected] RNA interference (RNAi) is a powerful technique through which double stranded RNA induces the activation of cellular pathways, leading to potent and selective silencing of genes with homology to the double strand. The ever-growing list of potential applications has contributed to rapid growth of this technique and to the understanding of its biology. Though biologically evolved to mediate resistance to both endogenous and exogenous pathogenic nucleic acids and to regulate the expression of protein coding genes, it is being successfully used as a tool for functional genomics and to manipulate the expression of desired genes. The initial suggestion of RNAi came from work in petunia flowers in which overexpression of the gene responsible for purple pigmentation actually caused the flowers to lose their endogenous colours. Antisense and sense suppression were described in petunia using the chalcone synthase (CHS) gene. Since then, flower colour biosynthesis has been down regulated through antisense or sense suppression in many ornamental plants, such as rose, carnation and chrysanthemum. Novel flower colour is industrially significant. Besides artificial modulations of flower colours are one of the important subjects of current plant biotechnology. This study aimed to explain that different flower colour modification studies using RNAi technique. doi:10.1016/j.copbio.2011.05.114
OP44 Development of an effective transformation system for the nuclear genome of the flagellate Euglena gracilis Juraj Krajcovic 1 , Vamsi K Vejerla 3 , Rostislav Vacula 1 , Eva Dobáková 1 , Gabriela Gavurníková 2 , Steven D Schwartzbach 3 1
Institute of Cell Biology and Biotechnology, FNS, Comenius University, Bratislava, Slovakia 2 Department of Biochemistry, FNS, Comenius University, Bratislava, Slovakia 3 Department of Biological Sciences, University of Memphis, Memphis, TN, USA E-mail address: [email protected] (J. Krajcovic) A method has been developed for nuclear genome transformation of the flagellate Euglena gracilis as part of a long-term project to identify the targeting domains of nuclear encoded plastid proteins. A zeocin resistance transformation cassette encoding the selectable zeocin resistant marker under control of the Euglena LHCPII promoter was constructed by inserting the Sh ble gene conferring zeocin resistance between the 5 and 3 end of the Euglena LHCPII gene. Zeocin resistant cells were isolated after electrophoretic or biolistic transformation. Three rounds of selection by growth with zeocin in liquid media followed by colony selection on
S45
plates were required to obtain resistant cells that no longer segregated the zeocin sensitive phenotype during subsequent growth. PCR demonstrated the presence of the Sh ble gene linked to the 5 and 3 end of the Euglena LHCPII gene in transformed cells. Expression of the Sh ble protein under control of the LHCPII promoter was demonstrated by western blotting. Cells maintained in culture for one year under nonselective conditions exhibited the zeocin resistant phenotype, contained the zeocin resistant gene as measured by PCR and expressed the Sh ble protein. These results indicate that stable transformation of Euglena has been achieved. doi:10.1016/j.copbio.2011.05.115
OP45 Differential expression of genes possibly involved in incompatible interaction between barley and powdery mildew Figen Ersoy 1 , Christopher J Ridout 2 , Mahinur S Akkaya 3 1
Biology Department, Uludag University, Bursa, Turkey Disease and Stress Biology, John Innes Centre, Norwich, UK 3 Department of Chemistry, Middle East Technical University, Ankara, Turkey 2
E-mail address: fi[email protected] (F. Ersoy) The various domains of a plant disease resistance protein from wheat were found to be interacting with yeast proteins when screened via yeast two hybrid analyses. These genes are considered to play roles in disease resistance response. Thus, the expression levels in Mla3 mediated Powdery Mildew (Blumeria graminis f.sp. hordei, Bgh) disease resistance in barley were determined. The barley homologs of ARD1, CPR7, CSE1, GCN2 and SRP72, were partially cloned and sequenced. Their differential expression was confirmed using qRT-RCR at 6 hpi, 12 hpi, 24 hpi and 72 hpi upon incompatible Bgh infection in a resistant barley line (Pallas-02). All of the genes except HvCPR7 showed maximum expression levels at 12 hpi and gradually dropped at 24 and 72 hpi. On the other hand, HvCPR7 showed highest induction at 6 hpi, indicating that HvCRP7 is an even earlier responding gene. Most of the human homologues of these genes have been widely studied and found to have roles in apoptosis. As apoptosis is very important in the plant disease resistance response, qRT-PCR was performed to check if the genes are induced in resistant barley after powdery mildew inoculation. All the five genes were found to be differentially expressed in incompatible interactions between barley and powdery mildew. doi:10.1016/j.copbio.2011.05.116
OP46 The effect of in vitro competition on callus formation and shoot regeneration from mature embryos of wheat (Triticum sp.) Seda Pelit 1 , Mustafa Yildiz 2 , Cansu Telci 2 , Burak Onol 2 1
Graduate School of Natural and Applied Sciences, University of Ankara, Ankara, Turkey 2 Faculty of Agriculture, University of Ankara, Ankara, Turkey E-mail address: [email protected] (M. Yildiz) Food demand of about two-thirds of the world population is based on wheat. Plants compete for light above ground with their leaves and shoots, and for water and nutrients under-ground with their roots. Plant density as a biotic stress factor in natural con-
that S. coronopifolius and A. armata produce biomolecules with high antitumoral potential. doi:10.1016/j.copbio.2011.05.113
OP43 Obtaining new flower colors by RNA interference technique Sebnem Kavakli Biotechnology Department, Graduate School of Natural and Applied Sciences, Ege University, Izmir, Turkey E-mail address: [email protected] RNA interference (RNAi) is a powerful technique through which double stranded RNA induces the activation of cellular pathways, leading to potent and selective silencing of genes with homology to the double strand. The ever-growing list of potential applications has contributed to rapid growth of this technique and to the understanding of its biology. Though biologically evolved to mediate resistance to both endogenous and exogenous pathogenic nucleic acids and to regulate the expression of protein coding genes, it is being successfully used as a tool for functional genomics and to manipulate the expression of desired genes. The initial suggestion of RNAi came from work in petunia flowers in which overexpression of the gene responsible for purple pigmentation actually caused the flowers to lose their endogenous colours. Antisense and sense suppression were described in petunia using the chalcone synthase (CHS) gene. Since then, flower colour biosynthesis has been down regulated through antisense or sense suppression in many ornamental plants, such as rose, carnation and chrysanthemum. Novel flower colour is industrially significant. Besides artificial modulations of flower colours are one of the important subjects of current plant biotechnology. This study aimed to explain that different flower colour modification studies using RNAi technique. doi:10.1016/j.copbio.2011.05.114
OP44 Development of an effective transformation system for the nuclear genome of the flagellate Euglena gracilis Juraj Krajcovic 1 , Vamsi K Vejerla 3 , Rostislav Vacula 1 , Eva Dobáková 1 , Gabriela Gavurníková 2 , Steven D Schwartzbach 3 1
Institute of Cell Biology and Biotechnology, FNS, Comenius University, Bratislava, Slovakia 2 Department of Biochemistry, FNS, Comenius University, Bratislava, Slovakia 3 Department of Biological Sciences, University of Memphis, Memphis, TN, USA E-mail address: [email protected] (J. Krajcovic) A method has been developed for nuclear genome transformation of the flagellate Euglena gracilis as part of a long-term project to identify the targeting domains of nuclear encoded plastid proteins. A zeocin resistance transformation cassette encoding the selectable zeocin resistant marker under control of the Euglena LHCPII promoter was constructed by inserting the Sh ble gene conferring zeocin resistance between the 5 and 3 end of the Euglena LHCPII gene. Zeocin resistant cells were isolated after electrophoretic or biolistic transformation. Three rounds of selection by growth with zeocin in liquid media followed by colony selection on
S45
plates were required to obtain resistant cells that no longer segregated the zeocin sensitive phenotype during subsequent growth. PCR demonstrated the presence of the Sh ble gene linked to the 5 and 3 end of the Euglena LHCPII gene in transformed cells. Expression of the Sh ble protein under control of the LHCPII promoter was demonstrated by western blotting. Cells maintained in culture for one year under nonselective conditions exhibited the zeocin resistant phenotype, contained the zeocin resistant gene as measured by PCR and expressed the Sh ble protein. These results indicate that stable transformation of Euglena has been achieved. doi:10.1016/j.copbio.2011.05.115
OP45 Differential expression of genes possibly involved in incompatible interaction between barley and powdery mildew Figen Ersoy 1 , Christopher J Ridout 2 , Mahinur S Akkaya 3 1
Biology Department, Uludag University, Bursa, Turkey Disease and Stress Biology, John Innes Centre, Norwich, UK 3 Department of Chemistry, Middle East Technical University, Ankara, Turkey 2
E-mail address: fi[email protected] (F. Ersoy) The various domains of a plant disease resistance protein from wheat were found to be interacting with yeast proteins when screened via yeast two hybrid analyses. These genes are considered to play roles in disease resistance response. Thus, the expression levels in Mla3 mediated Powdery Mildew (Blumeria graminis f.sp. hordei, Bgh) disease resistance in barley were determined. The barley homologs of ARD1, CPR7, CSE1, GCN2 and SRP72, were partially cloned and sequenced. Their differential expression was confirmed using qRT-RCR at 6 hpi, 12 hpi, 24 hpi and 72 hpi upon incompatible Bgh infection in a resistant barley line (Pallas-02). All of the genes except HvCPR7 showed maximum expression levels at 12 hpi and gradually dropped at 24 and 72 hpi. On the other hand, HvCPR7 showed highest induction at 6 hpi, indicating that HvCRP7 is an even earlier responding gene. Most of the human homologues of these genes have been widely studied and found to have roles in apoptosis. As apoptosis is very important in the plant disease resistance response, qRT-PCR was performed to check if the genes are induced in resistant barley after powdery mildew inoculation. All the five genes were found to be differentially expressed in incompatible interactions between barley and powdery mildew. doi:10.1016/j.copbio.2011.05.116
OP46 The effect of in vitro competition on callus formation and shoot regeneration from mature embryos of wheat (Triticum sp.) Seda Pelit 1 , Mustafa Yildiz 2 , Cansu Telci 2 , Burak Onol 2 1
Graduate School of Natural and Applied Sciences, University of Ankara, Ankara, Turkey 2 Faculty of Agriculture, University of Ankara, Ankara, Turkey E-mail address: [email protected] (M. Yildiz) Food demand of about two-thirds of the world population is based on wheat. Plants compete for light above ground with their leaves and shoots, and for water and nutrients under-ground with their roots. Plant density as a biotic stress factor in natural con-