Isolation Of Stolon Specific Expressing Gene And Acquisition Of Transgenic Lines For Regulation Of The Exprossion In Potato

In recent years, studies on cloning and functionality of genes related to organogenesis and development of plant have obtained a series of important achievements. However, most of the studies, started with a single gene, describe function of those genes and position them in the regulation network during organogenesis or development of plant. Therefore, molecular mechanism of organogenesis and development of plant is still in the early stage.Previous study isolated stolon specific-expressing EST N4S7and S36in our lab. In this study, RACE technique was employed to isolated full-length cDNA from which EST N4S7and S36were identified. Sequence analysis of the cDNA was carried out with bioinformatic tools to predict function of the cDNA. Vectors for expression in vitro and in vivo were constructed. Positive PCR outcome indicates that three cDNAs were transformed into potato. This makes it possible to reveal function of those cDNA in stolon development. Specific result of this study is as following:1.3full-length cDNAs, W2, W3-2and W3were obtained with the RACE. BLAST with cDNAs indicated that W2was100%similar to a cDNA sequence from tobacco mitochondria, which encodes NADH dehydrogenase subunit-4. W3-2shares77%similarity to a cDNA encoding ataxin-3in Soybean. W3has95%similarity to a cDNA encoding the DnaJ in the potato. Therefore, we predict functions of W2, W3-2and W3corresponding as described above.2. With W2, W3-2, two recombining plasmids, pET-28c-W2and pET-28c-W3-2, constructed and transformed into Escherichia coli BL21(DE3). Growth curves of two recombining strains were assayed. The result showed that there is no significant difference between recombining strains and control before and after IPTG inducing. It suggests that expression of two cDNA in the host cells does not generate effect to the host growth.3. With W2, W3-2and W3, transformation vectors, either over-expressing or knockout-expression of target genes were constructed. Through agrobacterium-medium approach, sense and antisense cDNA of W2, W3-2and W3were transformed into potato. Positive PCR indicated that three cDNAs were fused into potato genome. Transgenic lines make it possible to reveal function of those cDNA in stolon development.