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内容記述 |
As a major provision for more than half of the world’s population, rice (<i>Oryza sativa</i> 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 <i>Poaceae</i> 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 <i>Agrobacterium tumefaciens</i>-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, <i>nDart1-0 (<u>n</u>on-autonomous <u>D</u>NA-based <u>a</u>ctive <u>r</u>ice <u>t</u>ransposon one-zero</i>) in the <i>hAT</i> superfamily, had been identified as a causative element of spontaneous leaf variegation shown in the mutant line pyl-v (<u>p</u>ale-<u>y</u>ellow-<u>l</u>eaf <u>v</u>ariegated). This mutable phenotype is caused by the disruption and restoration of the nuclear-coded essential chloroplast protease gene, <i>OsClpP5</i>, due to the insertion and subsequent excision of <i>nDart1-0</i>. As a typical non-autonomous transposon in the <i>hAT</i><br/>superfamily, <i>nDart1-0</i> can transpose only when the <i>trans</i>-acting transposase is supplied from an autonomous element, <i>aDart (<u>a</u>ctive autonomous <u>Dart</u></i>). On the other hand, an indicator line, pyl-stb (<u>pyl</u>-<u>st</u>a<u>b</u>le) shows uniform pale-yellow leaves with no <i>nDart1-0</i> excision due to a lack of an <i>aDart</i>. The result of test crosses between pyl-v and pyl-stb lines indicated that the pyl-v line carries an <i>aDart</i> element in its genome.<br /> In the published genomic sequence of the cultivar Nipponbare, there are 38 candidate autonomous <i>Dart</i> 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 <i>aDart</i> elements in its genome. Meanwhile, the excision of some endogenous <i>nDart1</i> 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, <i>iDarts</i> (<i>inactive autonomous <u>Darts</u></i>).<br /> The first aims of this study were identifying the <i>aDart</i> 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 <i>aDart</i> element in the pyl-v line coincides with one of the 38 candidate autonomous elements, <i>iDart1-27</i>, residing on chromosome 6 in Nipponbare. Also, I have found that all of the examined transcripts of the <i>Dart</i> transposase gene were derived from <i>Dart1-27</i>in the pyl-v line. These results strongly suggested that <i>Dart1-27</i>in pyl-v acts on <i>nDart1-0</i> as an active <i>aDart</i> element. Then, I demonstrated that <i>iDart1-27</i> cloned from the Nipponbare genome can be converted to an active <i>aDart</i> element in <i>Arabidopsis thaliana</i> plants when its methylation status was eliminated during the cloning process; <i>Dart1-27</i> excised <i>nDart1-0</i> as well as itself from the introduced vectors and integrated into various sites of the <i>A. thaliana</i>genome. These results clearly indicated that <i>Dart1-27</i> is a functional autonomous element, and it is active as an <i>aDart</i> element in the pyl-v line whereas epigenetically silenced as <i>iDart1- 27</i> in Nipponbare. Furthermore, I showed other <i>Dart</i> elements, <i>Dart1-1, Dart1-20, Dart1-28</i> and <i>Dart1-52</i> are also functional autonomous elements, but they are epigenetically silenced as <i>iDarts</i> in Nipponbare.<br /> Next, in order to study if there are any regulatory mechanisms that control the activity of the <i>Dart/nDart</i> system in the pyl-stb line, I introduced <i>Dart1-27</i> derivatives into the pyl-stb line and evaluated their activity. As a prerequisite for this transgenic approach, I carefully confirmed that during each step of the <i>A. tumefaciens</i>-mediated transformation process the endogenous <i>iDart</i> elements in the pyl-stb genome are almost never activated (0.1%). Based on this confirmation, I introduced <i>Dart1-27</i> derivatives into pyl-stb and demonstrated that they can mobilize <i>nDart1-0</i> elements from the <i>OsClpP5</i> gene as well as from an introduced <i>GUSPlus</i> gene at a high frequency in transgenic pyl-stb plants. This result reconfirmed that <i>Dart1-27</i> is a functional autonomous element able to act on <i>nDart</i> elements when its methylation status is eliminated, as shown in <i>A. thaliana</i>plants. From the results of phenotypic analysis of transgenic pyl-stb plants, it was suggested that there is a development-dependent regulation of <i>Dart</i>activity in regenerated pyl-stb plants; most of the transgenic pyl-stb plants introduced with <i>Dart1-27</i> derivatives were the pyl-stb phenotype at their 4-6 leaves stage, but almost all of them became the pyl-v phenotype at their 7-10 leaves stage.<br /> In this manuscript, I have unambiguously demonstrated that the active autonomous element in the pyl-v line is <i>Dart1-27</i> on chromosome 6 and that the rice genome contains multiple potential autonomous <i>Dart</i> elements silenced epigenetically. From analysis of transgenic pyl-stb plants, I have also indicated a development-dependent regulation that could be a key to further elucidating <i>Dart/nDart</i> regulation mechanisms in the rice genome. I believe these results will facilitate an effective gene tagging system using the <i>Dart/nDart</i> elements in rice. |
要旨・審査要旨 (344.0 kB)
