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1

RNAi as a tool for functional genomics

100%
Kisiel A., Podkowinski J., Figlerowicz M.
Biotechnologia
|
2003
|
issue 2
104-119
EN
Sequencing eukariotic genomes generates a large amount of sequence data. There is an urgent need for developing fast and effective methods of functional analysis of newly discovered genes. Postranscriptional gene silencing can be applied for functional genomics of plants and animals. A large scale analysis of C. elegans genome has been already performed. For mammals and plants, current work focuses on constructing high-throughput silencing vectors.
2

RNAi, PTGS and quelling - three variations on one subject?

80%
Szweykowska-Kulinska Z., Jarmolowski A., Figlerowicz M.
|
EN
In 1990, it was already discovered that plant transformation with a transgene containing its homologue in the plant nuclear genome is able to promote silencing of both the transgene and the homologous, endogenous gene. The phenomenon was named posttranscriptional gene silencing (PTGS) or co-suppression. The same results were obtained when a transgene was introduced into the nuclear genome of fungus Neurospora crassa. This process was termed quelling. In 1998, RNAi (RNA interference) was discovered in the Caenorhabditis elegans worm. Specific gene silencing occurred after the introduction into the worm of cells of double stranded RNA with sequence complementarity to the endogenous gene. It was shown that RNAi operates at the stage of the mature mRNA in the cytoplasm. dsRNAs are converted into siRNAs (small interfering RNAs) due to the Dicer enzyme activity. siRNAs are incorporated into the RISC (RNA ? induced silencing complex). Active RISC promotes specific mRNA degradation. RNAi/PTGS/quelling processes show many mechanistic similarities, but they also differ in some details. All of them represent an ancient mechanism that probably evolved to protect eukaryotic cells against invasive forms of nucleic acids like viruses, transposons, and others.
3

Application of RNA interference for cereal crops biotechnology

70%
Nadolska-Orczyk A., Zalewski W., Gasparis S., Orczyk W.
Biotechnologia
|
2010
|
issue 3
53-63
EN
RNAi technology is based on a natural process of RNA-directed gene regulation. The technique is widely used for gene functional analysis and to obtain plants with modified traits. The main advantage of this system, particularly when applied for polyploid species, is the possibility of simultaneous silencing of homologous, homoeologous or orthologous genes. The article discusses the results of relatively few papers where RNAi has been used for functional analysis of native genes of wheat and barley. The main part of the article presents the research on RNAi based gene silencing in cereals performed by our group. The experimental basis of our work was the elaboration of efficient Agrobacterium-based transformation and plant regeneration systems of different cereal species (wheat, barley, triticale and oat). Currently, the method is applied for modification of two types of traits in wheat, triticale and barley. The first one is a technological trait related to cereal grain hardness. It is genetically controlled by Pina and Pinb genes. We obtained over a hundred transgenic lines with various degrees of Pina and Pinb silencing. Currently, the lines are being analyzed for the amount of PINA and PINB proteins, composition of storage proteins, and the grain texture. The second set of traits depends on CKX genes encoding cytokinin oxidase/dehydrogenase ? the part of the system specifically governing the cytokinin level in different organs and developmental stages. We obtained over forty barley transgenic lines with silenced HvCKX1. This modification was found to be tightly correlated with enhanced plant productivity measured as the higher grain number and higher mass of a thousand kernels. The T1 and T2 transgenic seedlings developed bigger root system.
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