Genetic Transformation Of AmiRNA Interference Vectors Targeted For Homologous Genes (rps7, Ccb256, Cox3) Between Nuclear And Mitochondrial Genomes In Arabidopsis Thaliana

Chloroplast and mitochondria are two most important organelles for energymetabolism, both of which have protist-originated and maternal-inherited genomes.Nucleus, chloroplast and mitochondria carry all the inheritance message of plants, sochloroplast and mitochondria are also very important in inheritance and evolution researchof plants. With the progress of sequencing projects and genome research, more and moreevidence showed that there exists transfer and exchange of inheritance message amongthree groups of genomes in plants. In this way, it makes great sense to study themechanism of coordinated regulation of certain genes between nucleus and organelles,find evidence and principle of DNA transfer, for it can be quite useful in trailing theevolution process of nucleus and organelles, and predicting their future.In this study, we integrate bioinformatics methods and gene engineering together.First we select homologous genes between nuclear and organelle genomes as the target.Then we try to interfere them by artificial microRNA gene silencing technology. As aresult, the mechanism of coordinated regulation of these genes between nucleus andorganelles would be revealed and gene transfer direction would also be clear. Theconclusion we got would supply several references for predicting the future evolutiondirection of organelles in plants. The main results are as follows:(1) Using BLAST method, we found rps7、ccb256、cox3in nuclear genome ofA.thaliana as the targets, which have homologous copies in mitochondrial genome, andeach of them has just one copy in nuclear genome. Then we designed amiRNA for thesegenes with the help of WMD3.(2) Silencing vectors were transformed into A.thaliana mediated by Agrobacterium.Seeds of T0generation were screened by hygromycin. Nine positive transformants wereobtained through PCR confirmation. (3) RT-PCR results of three transformants survived showed that the expression ofccb256gene in nucleus was totally inhibited, while the expression of rps7gene wassignificantly enhanced. No result of cox3gene was got for none of its transformantssurvived.Concerning the gene function of rps7and ccb256, we came to a conclusion thatccb256gene is mainly regulated by mitochondrial genome, and maybe it ismitochondrial-originated, then a copy was given to nuclear genome. rps7gene is mainlyregulated by nuclear genome. In the same way we may conclude that it isnuclear-originated, so its homologous gene in mitochondrial genome is just a copy. Theconclusion we drew is that there really exists bidirectional DNA exchange betweennucleus and mitochondria, however, the future evolution direction of mitochondria is stillhard to predict.The results of present research has paved the way for studying the principle of DNAtransfer among nuclear, chloroplast and mitochondrial genomes, as well as for predictingthe future evolution direction of organelle genomes.