Characterization of autonomous Dart1　transposons belonging to the hAT superfamily in rice

As a major provision for more than half of the world’s population, rice (Oryza sativa L.) is one of the most important plant species and has great economic importance. Also, rice has been focused on as an excellent model plant for cereal genomics studies due to its following features; 1) the smallest genome size (389 Mb) among the cereal grasses, 2) syntenic relation with other agronomically important Poaceae species such as maize, barley and wheat, 3) available varied resources including a large number of genetic markers, genomic libraries, many mutant lines and retrotransposon tagged lines, 4) there exists well developed technologies for rice genome manipulation such as Agrobacterium tumefaciens-mediated gene transformation and gene targeting with homologous recombination. Consequently, fine quality map-based genome sequencing of rice was completed in 2005.<br />　　One of the most challenging goals for the plant-research community going forward is to identify the function and regulation of rice genes. Thus, both forward and reverse genetic approaches have been developed to elucidate these functions. However, because the tissue culture process is a necessary step in most of the currently available procedures used in rice genome research, somaclonal variations, which refer to genetic and epigenetic mutations induced by tissue culture, can hamper these approaches. Therefore, a lot of attention has been given to recently identified endogenous DNA transposons that are active under natural growth conditions, a character that is quite useful to development of more efficient rice transposon tagging as a functional genomics tool free from somaclonal variation.<br />　　One such DNA transposon, nDart1-0 (non-autonomous DNA-based active rice transposon one-zero) in the hAT superfamily, had been identified as a causative element of spontaneous leaf variegation shown in the mutant line pyl-v (pale-yellow-leaf variegated). This mutable phenotype is caused by the disruption and restoration of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to the insertion and subsequent excision of nDart1-0. As a typical non-autonomous transposon in the hAT<br/>superfamily, nDart1-0 can transpose only when the trans-acting transposase is supplied from an autonomous element, aDart (active autonomous Dart). On the other hand, an indicator line, pyl-stb (pyl-stable) shows uniform pale-yellow leaves with no nDart1-0 excision due to a lack of an aDart. The result of test crosses between pyl-v and pyl-stb lines indicated that the pyl-v line carries an aDart element in its genome.<br />　　In the published genomic sequence of the cultivar Nipponbare, there are 38 candidate autonomous Dart elements that have putative transposase genes with no apparent nonsense or frameshift mutations. However, from the result of test crosses with the pyl -stb line, it was shown that Nipponbare carries no aDart elements in its genome. Meanwhile, the excision of some endogenous nDart1 elements in Nipponbare and pyl- stb was induced by treatment with a DNA methylation inhibitor, 5-azacytidine. Hence, these lines were predicted to carry epigenetically silenced autonomous elements, iDarts (inactive autonomous Darts).<br />　　The first aims of this study were identifying the aDart element in the pyl-v line and demonstrating its molecular criteria as an autonomous element. To this end, I performed map-based cloning and revealed that the aDart element in the pyl-v line coincides with one of the 38 candidate autonomous elements, iDart1-27, residing on chromosome 6 in Nipponbare. Also, I have found that all of the examined transcripts of the Dart transposase gene were derived from Dart1-27in the pyl-v line. These results strongly suggested that Dart1-27in pyl-v acts on nDart1-0 as an active aDart element. Then, I demonstrated that iDart1-27 cloned from the Nipponbare genome can be converted to an active aDart element in Arabidopsis thaliana plants when its methylation status was eliminated during the cloning process; Dart1-27 excised nDart1-0 as well as itself from the introduced vectors and integrated into various sites of the A. thalianagenome. These results clearly indicated that Dart1-27 is a functional autonomous element, and it is active as an aDart element in the pyl-v line whereas epigenetically silenced as iDart1- 27 in Nipponbare. Furthermore, I showed other Dart elements, Dart1-1, Dart1-20, Dart1-28 and Dart1-52 are also functional autonomous elements, but they are epigenetically silenced as iDarts in Nipponbare.<br /> 　Next, in order to study if there are ...

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