Experimental data suggest that four billion years ago amino acids, purines and pyrimidines could be present in prebiotic soup. Non- enzymatic synthesis of peptides and RNA is possible. RNA can act as replicators and catalysts. Ribonucleic acids are very likely to be recognized as the molecules that gave rise to life. Riboorganisms used amino acids for the synthesis of purine and pyrimidine rings. They developed genetic code and translation. First proteins were used to create channels across phospholipid membranes and to stabilize ribozymes. Ribonucleoproteins containing catalytic RNA can be regarded as relicts from RNA World.
The challenge for the plant functional genomics is to elucidate the function of every gene in the plant genome. Definitive allocation of gene function requires the introduction of mutations and further analysis of their phenotypic repercussions. Chemical and ionizing radiation mutagenesis are useful tools to generate genetic variability for functional genomic studies. T-DNA fragments and transposable elements can potentially generate mutations disrupting gene continuity. ZFN mutagenesis method allows the introduction of genetic changes precisely into the desired genome region.
Gene silencing and modeling of gene expression are classical approaches in studying gene functions. There are many methods available which use RNA molecules as gene silencing inducers or gene expression modulators. RNA molecules act at different levels of gene expression: chromatin structure, gene transcription and pre-mRNA maturation. In the last decade, new methods for silencing and modeling of gene expression emerged, utilizing RNAi phenomenon, trans-splicing, RNA directed DNA metylation, ribozymes, artificial microRNAs, ryboswitches, and U1 interference. In this paper, we review some of the methods which were successfully used for gene function studies and as therapeutic tools against different plant and human diseases.
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.
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