| Gibberellins(GAs) are important plant hormones, involved in various developmental and physiological processes of plants.GA20 oxidase is the key regulatory enzyme in GA biosynthesis and metabolism pathway. Cloning and studying the function of GA20 oxidase gene from turfgrass was a basic work for turfgrass dwarf breeding.The GA20 oxidase was obtained from wild Zoysiagrass(Zoysia japonica steud.) by the means of RT-PCR and RACE, the pET-30a-GA20 vector, pC35S1303GA20+and pC35S1303GA20- vectors, pC1303GA20+ and pC1303GA20- vectors were constructed respectively. The expression of GA20 in E.Coli, tobacco, tall fescue (Festuca arundinacea L.) and zoysiagrass were discussed and the conclusions showed as followings:1, A pair of degenerate primers were designed according to the conserved sequence of GA20 oxidase of several gramineous plants the GA20 oxidase was isolated from wild Zoysiagrass by the method of RT-PCR and RACE. The gene cDNA full-length was 1311bp, containing a 1176 bp open reading frame, encoding a putative polypeptide of 392 amino acids. The GA20 oxidase in Zoysiagrass shared 55%-73% homology with wheat, barley, Oryza sativa and Lolium perenne. The GenBank accession number of GA20 oxidase gene isolated from Zoysiagrass is DQ645453.2, The GA20 oxidase gene was cloned into the prokaryotic expression vector pET-30a and expressed in E. Coli BL21(DE3) strain under induced 1h, 2h, 3h, 4h and 5h by IPTG The product was identified by SDS-PAGE and the results showed that the specific expression of 45kDa protein was achieved, and the protein weight was increasing with the induce time increasing. 3, The restriction eddonuclease BamH I and BisW I were used to construct sense expression vector pC35S1303GA20+ and antisense expression vector pC35S1303GA20- adapted to dicotyledonous plants genetic transformation. Ttansgenic tobacco plants obtained from leaf discs applying Agrobacteriummediated gene transfer carrying pC35S1303GA20+ and pC35S1303GA20-. 28 individuals hygromycin-resistant plants were recovered involving in 12 individuals transformed pC35S1303GA20+ and 16 individual transformed pC35S1303GA20- hygromycin-resistant plants. Hygromycin-resistant plants were identified by PCR and PCR-Southern blotting respectively, 8 individuals with pC35S1303GA20+ were obtained and 11 individuals with pC35S1303GA20-were obtained. The putative transgenic plants frequency was 67.8%. Transgenic tobacco growthrate was detected. As a whole, the growth rate faster degree as follows: pC35S1303GA20+ transgenic plants >control > pC35S1303GA20- transgenic plants. The root quantity of pC35S1303GA20+ transgenic plants was less than that of pC35S1303GA20- transgenic plants. These pC35S1303GA20+ transgenic plants displayed a phenotype that may be attributed to the overproduction of GA. The phenotype included a longer hypocotyl, less and lighter-green leaves, increased stem elongation and slimmer, and pC35S1303GA20- transgenic plants displayed a phenotype that may be attributed to the reducing production of GA, the phenotype included stem turned shorter and thicker, larger and darker-green leaves.4, The restriction eddonuclease BamH I and BisW I were used to construct sense expression vector pC1303GA20+ and antisense expression vector pC31303GA20- adapted to monocotyledon plants genetic transformation. Plant regeneration system and Biolistic gene deliver system constructed by our laboratory was used in the experiments. Embryonic callus of tall fescue and zoysiagrass were used as material for genetic transformation with the sense and antisense GA20 oxidase gene by particle bombardment. The transgenic plants were obtained. Through PCR and PCR-Southern blot analysis, 2 zoysiagrass individual and 39 individuals tall fescue transformed pC1303GA20+ were obtained, including in Barlexus 35 individuals and Millennium 4 individuals; 30 individuals tall fescue transformed pC1303GA20- were obtained, including in Barlexus 27 individuals and Millennium3 individuals. There was not remarkbale dissimilarity in the phenotype between pC1303GA20- transgenic of zoysiagrass and control. Tall fescue pC35S1303GA20+ transgenic plants displayed a phenotype that may be attributed to the overproduction of GA. The phenotype included a longer hypocotyl, less and lighter-green leaves, increased stem elongation and slimmer. And tall fescue pC35S1303GA20- transgenic plants displayed a phenotype that may be attributed to the reducing production of GA, the phenotype included stem turned shorter and thicker, larger and darker-green leaves. |
