Specific Gene Silencing Of DUF647 Family Genes In Arabidopsis By Artificial MicroRNAs

DUF647 gene family, which comprises genes ATIG13770, AT2G23470, AT2G31190, AT3G45890, AT5G01510 and AT5G49820, is an unknown function gene family in Arabidopsis genome. The mutants of AT2G31190 and AT3G45890 had been obtained by EMS mutagenesis, but AT1G13770, AT2G23470, AT5G01510 and AT5G49820 knockout mutants are not yet available. Artificial microRNAs (amiRNAs) provide a new and highly specific approach for effective post-transcriptional gene silencing in plants. This thesis aims to specific gene silencing of AT1G13770, AT2G23470, AT5G01510 and AT5G49820 by amiRNAs.We designed four amiRNAs targeting four genes (AT1G13770, AT2G23470, AT5G01510, AT5G49820) respectively by using the WMD (Web MicroRNA Designer) platform. The 21mers sequence was engineered into MIR319a precursor using overlap PCR to replace the endogenous miRNA sequence. The amiRNA backbone was transferred into binary vector pCHF3. The conformed plasmids that harbor amiRNA stemloop fragments driven by CaM35S promoter were introduced into Agrobacterium strain AGL1 and subsequently transformed into Arabidopsis Col background plants. Transgenic Arabidopsis lines harboring the overexpression constructs showed an efficient knockdown of the target mRNAs. Both the vegetative and reproductive organs of transgenic lines show developmental defects, especially AT1G13770-amiRNA and AT5G49820-amiRNA. These results suggest that AT1G13770, AT2G23470, AT5G01510 and AT5G49820 play key roles in Arabidopsis developmental processes.