Establishment And Application Of High Throughput Transformation Systems In Rice

Plant transformation is a powerful tool for orop improvement and funotional genomics study.Although many improvements have been made to increase the transformation efficiency and coverage of plant species in the past twenty years,there are still plant species or genotypes which are recalcitrant for transformation.It is of great interest to develop new technologies to further improve transformation efficiency in plants.In this study,the effect of H2A1 gene,a plant gene involving in the plant-Agrobacterium interaction,on efficiency of Agrobacterium-mediated transformation was evaluated in rice.In addition to that,we also investigated the effectiveness of the high throughput RNAi vectors based on Gateway^TM(Invitrogen) technology by functional analysis of genes participating in iron homeostasis in rice.Results are summarized as follows:1.Using Nipponbare(Oryza sativa ssp.Japonica)as the recipient,the function of Arabidopsis thaliana gene H2A-1(AtHTA1)encoding a histone H2A-1 protein was investigated.The re-transformation efficiency of T1 generation transformants overexpressing of H2A-1 increased for 44%compared to the empty vector controls.2.Using Gateway^TMtechnology,7 RNA interference vectors were constructed to knockdown genes involving in iron homeostasis in wildtype and the naat1 mutant(mutation in nicotiamine transferase)rice.The target genes include rice nicotiamine synthase OsNAS1,OsNAS2,and OsNAS3,iron-regulated metal transporters OsIRT1 and OsIRT2.RT-PCR analysis on the T1 transformants showed that the introduction of the RNAi vectors suppressed the expression levels of the target genes to certain degrees.Suppression of OsNAS1,OsNAS2 simultaneously restored the phenotypes of naatl mutant. The finding will be an important part of the overall function of NAS in rice.