RNAi and Plant Gene Function Analysis Methods and Protocols
The use of RNAi technology is essential for most plant science researchers. As DNA sequence information increases, so the need for functional annotation of target genes also increases. Authoritative and accessible, RNAi and Plant Gene Function Analysis: M
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1. Introduction In the last decade, the genome sequences of some plants such as Arabidopsis, rice, poplar, papaya, soybean, and tomato have been made available. At the same time, we began to understand the function of small RNAs (sRNAs) of 19–25 nt in fine-tuning gene regulation in organisms. The system in which sRNAs suppress expression of specific genes based on sequence specificities is known as RNA interference (RNAi). sRNAs do not occur by chance but are actively produced for normal developmental progression or stress resistance of the organism. Now that we can decipher a rough map of RNAi mechanisms at the molecular level, we are at the stage where we can utilize this mechanism to regulate or modify some biological activities to improve plants or crops. Many different approaches are discussed in this H. Kodama, A. Komamine (eds.), RNAi and Plant Gene Function Analysis, Methods in Molecular Biology 744, DOI 10.1007/978-1-61779-123-9_1, © Springer Science+Business Media, LLC 2011
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book. In this chapter, I briefly overview the historical flow of research on RNAi over the last decade.
2. Discovery of RNA Silencing 2.1. Discovery of PTGS Events
During the long history of the plant sciences, many researchers have attempted to enhance the biological activities of plants. Soon after the establishment of the Agrobacterium-mediated transformation system to introduce foreign DNA, a cDNA copy of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco plants. Since wild-type plants do not have the CP gene, the transformed plants were forced to accommodate a novel foreign gene from outside their original genome. When the transformants were attacked by TMV, they showed less severe or delayed symptoms compared with the wild-type plants (1). In other words, the transformed plants showed some resistance against TMV and were described as immune to TMV infection. After screening populations of transformants, researchers identified good breeding resources that conferred satisfactory resistance upon crops, even those growing in field conditions. After this finding, several analogous approaches were applied in various host–virus systems. If a severe crop loss occurred due to infection by a certain virus, researchers determined the cause of the disease, identified the viral pathogen, made a cDNA of the virus, and determined its genome sequence. Based on available databases of virus information, they then assigned coding regions of the CP, replication protein, or movement protein in the genome. The CP coding region was then cloned and placed just downstream the cauliflower mosaic virus 35S promoter in a binary vector, and the crop plant of interest was transformed with the vector. Such transgenic plants showed less severe or delayed symptoms, and this newly acquired resistance could be expected to last for a year or two. This approach was very attractive, since conventional breeding techniques are time consuming and laborious to attain such traits to a similar level. The CP-mediated res
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