Cloning Of The GA Biosynthesis Genes: Ent-kaurene Oxidase & 20-oxidase And Fundamental Analysis Of Their Expression Modes In Fruit Trees

Gibberellins play multiple important roles in plant development. First they can regulate stem elongation, second they can induce flowering by act with the LEAFY promoter and they also play important role during seeds development and thus in the fruits development. And regulation of stem elongation, flower inducement and seeds and fruit development are three very crucial sections in fruit industrial, so it is important to clone the gibberellins biosynthetic enzyme genes and to research on their expressing modes and their regulation roles.In this article we cloned two crucial gibberellins biosynthesis enzyme genes: ent-kaurene oxidase and 20-oxidase, and we investigated their expression patterns.1 . We designed degenerate primers of consensus amino acid sequences of ent-kaurene oxdase, and use them to amply the middle segments of apple, strawberry and peach, and then we designed specific primers insides these middle segments to amplify 3' termini, and 5' termini. Finally we got the full length of strawberry and 3'teminal of peach. The full length of the strawberry ent-kaurene oxidase has 1650 bp , and code region has 539aa.The similarity among three rose family specy are 65%,79%,57%.2 . we cloned two ent-kaurene oxidase homologues from the reverse transcriptional products of different strawberry tissue, The identity of these two homologues is 88.2%. It suggest us that it belongs to a gene family.3. By investigate the primary structure of strawberry ent-kaurene oxidase ,we found that it has four conserve amino acid sequence. It suggested that these conserved regions are important in gene function. To investigate the secondary structure of this gene, we found it has a transmembrane region near the N-terminus, followed by a proline- rich conserved region,and it has a conserved haemachrome binding region about 60 aa away from carboxyl terminus.4. We use half quantification and digoxigenin detection to investigate the expression pattern of ent-kaurene oxidase. We found that it was regulated by development, it expressed high in the fast growing tissue , expanded leaves under a fast growing circumstance also express high level of ent-kaurene oxidase. The expression level increase during the vigorous growing period.5. 20-oxidase in charged of transform inactive GAs to active GAs. Use Northern Blot ,it was only detected in developing seeds. We use RT-PCR to amplify theexpressing signal , and we found it can be detected in growthing leaves and stems. It offer us a worthwhile methods to detect and quantify low expressing gene.6. 20-oxidase expressed very low in expanded leaves of apple, it suggest that the GA intermediates synthesis by the expanded leaves may transport to other vigorous growing tissue as raw material for those tissue.7. we found ent-kaurene oxdase belong to a multigene family by southern blot .we deduced that multigene response to different development signal.