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Expression Of RNAi Construct With Two Rice Methyltransferases 1-1 And 1-2 Genes In Japonica Rice

Posted on:2010-03-01Degree:MasterType:Thesis
Country:ChinaCandidate:Y X LuFull Text:PDF
GTID:2120360275489203Subject:Botany
Abstract/Summary:PDF Full Text Request
Cytosine methylation is an epigenetic process that mediates the gene expression during different development stages in special tissues. Cytosine methylation is carried out by DNA methyltransferases (DNA MTases), which can transfer a methyl group from S-adenosyl methionine to cytosine residues of the double helix, a common phenominium found in both prokaryotes and eukaryotes. The accurate functions of methylation in plants are still not evidentially known. In this paper, both OsMET1-1and OsMET1-2 are silenced at the same time by RNA interference technology to study the functions of methylation in plants. We intend to investigate the further function of methylation in plants after phenotype identification and molecular examinations, which can supply some basis to explain the reason and mechanism of methylation in epigenic mutation.The recombinant vector expressing two methyltransferases 1-1 and 1-2-targeted genes with short hairpin RNA of Oryza sativa was constructed. Two pairs of DNA sequences were designed and then combined into complementary chains by amplification, respectively. The inverted sequence was first inserted into pSP72 and then to another side of intron in plasmid pKannibal to construct the recombinant vector after enzyme digestion by XhoI&KpnI. The recombinant vector was transformed into E. coli strain DH5αfor screening and identifying. The agarose electrophoresis and sequencing analysis showed that the correct recombinant vector was obtained.In this study, the mature embryo-derived calli from two japonica cultivars (Songqian and 124) were chosen for use. Later, we optimized different factors affecting transformation and established a highly efficient transformatiom system. The transformation was performed with A. tumefaciens strain LBA4404 harboring the plasmid pBI121 using GFP as the reporter gene. Finally, we established a high frequency regeneration system for rice, defined callus induction medium, subculture medium and regeneration medium. Besides,factors such as the subculture time, the selection of medium, the concentration of A. tumefaciens, the time of co-culture and the drying treatment that affected the efficiency of transformion were also analyzed.Until now, we have obtained ten transgenic plants together, among which there are six Songqian and four 124 crops. GFP detection and PCR assay indicated that the target genes had been integrated into the rice genome. All the six Songqian rice have set seeds, but none have been got from four 124. The reason for that might be the species difference. Moreover, the transgenic plants have less tillers, seeds and curlier leaves, bloom earlier and have shorter growth period with comparison to plants from direct regeneration by tissue culture in phenotype. And the transgenic plants of 124 showed highly sterile besides those.The T1-generation seeds have been obtained and already seeded. Further measurements and study are being performed. These genetically-modified materials will be analyzed and subcultureed, which can provide some basis for investigating the further role of methylation in plant growth, plant development and epigenetics.
Keywords/Search Tags:Epigenetic, Methylation, RNAi, Rice, Genetic transformation
PDF Full Text Request
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