| Organ abscission is a physiological process that plant organs(flower,leaf,fruit etc.) separate from the parent plant. Abscission generally occurs in a predictable,specific area-abscission zone. Activation of abscission zone has always been the focus of plant organs abscission. IDA gene is essential for activation of abscission zone. So far, clear signal transduction pathways of organs abscission in Arabidopsis is Bopl/Bop2→ IDA→HAE/HSL2→MKK4/MKK5→MAPK→flower organs abscission.IDAin Arabidopsis, tomato,tobacco and soybean have been cloned and corresponding functional identified, but the gene was not reported in Petunia hybrida. In this experiment,IDA has been successfully separated and cloned(which is named PhIDL) from the flowers of petunia.The main results are as follows:1.We designed primers for the known sequences in EST of petunia. The PhIDL gene has been isolated and cloned with RT-PCR technology. This gene has total length of 381 bp,with a 270 complete open reading frame, encoding 89 amino acids. Full-length sequence also has 15 bp of 5’untranslated region and 94 bp of 3’untranslated region. Sequence alignment with other species such as tabacoo(Nicotiana tomentosiformis), tomato(Solanum lycopersicum),soybean(Glycine wax),western balsam poplar(Populus trichocarpa) in NCBI showed that 62%,49%,40%,42% sequence identity respectively with similar length of amino cncoded by the open reading frame.2.The molecular formula of PhIDL protein is C436H681N111O127S10,the theoretical molecular mass is 9830.4,isoelectric point(pl) is 8.84,the total average hydrophilic (GRAVY) is -0.199,the instability index is 59.05,this protein is a stable protein. The hydrophobic maximum is 2.8(24 amino acids),the minimum is -2.411(58 amino acids),15-35 amino acid is the obvious hydrophobic region. The protein has a transmembrane reigon,is in about 15-34 amino acids, commensurate with the hydrophobic region. The subcelluar localization of the protein is in nuclear. Secondary structure analysis of the protein indicates that the protein is a mixed protein with 32.58% a-helix,15.73% extended strand,6.74% beta turn and 44.94% of irregular coil, the protein is a mixed protein. The protein is a non-secretory type protein without signal peptide and has 6 phosphorylation sites.3.We have successfully constructed the plant binary expression vector PhIDL-p2355 and transformed the recombinant plasmids into Agrobacterium GV3101 with freeze-thaw method. After transformation of wild-type tobacco and Arabidopsis through Agrobacterium-mediated genetic, we have successfully got transgenic plants with resistance screening and PCR identification. |
