| Chemical inducible expression system is a powerful tool with which to regulate and control the gene expression of plant potentially in both laboratory and field. Their utility is dependent on the specificity and controllability of induction ofgene expression, as well as the cost and environment profile of the inducing chemicals. Searching new regulable gene by chemical is a basic work for the research and development of inducible promoter. In this study, a fungicide and two herbicide safeners were used as the inducer to treat the rice seedlings, and ten genes were isolated and characterized by differential display as the positive ones induced by the inducers. Analysis about the function and transcript element were carried out, and constructed plant were prepared. The following are the main result:1. Rice seedlings were induced by 3 inducers (Propiconazol,AD67,dichlopronamide) and expression analysis were carried out. Treatments were conducted at 3-1eaf stage, the samples were collected at 6h, 12h, 1d, 2d, 3d, 4d and 5d after induced and compared with the CK which treated by sterile water. Total RNA were extracted and differential display analysis show that 67 fragments were increased or decreased apparently in transcription level after induced.2. Fragments on the poly-gel were recycled and verified by reverse Northern blotting,ten positive fragments were identified as induced gene.Recycled DNA respond to induced gene were linked into T-vector and transferred to complementary cell of DH5α, after resistant screening, the positive clones were prepared for sequencing.3. Sequencing and homologous analyzing were processed for the 10 fragments.The searching on GeneBank revealed that fragments Y2 and Y11 come from the unknown sequence, and 2 fragments (Y1 and Y7) represent the unknown genes, the rest fragments respond to GMPase, Sm-F, RBCL, yippee, Vps55 and GRP/A respectively. 4. The transcript elements of 8 genes were searched in the 5' untranslation region. Multiple elements were founded in the 5' UTR, including the TATA-boxes as well as CAAT-boxes and G-box which is reserve in plant. And serial inducible elements such as ABRE which is respond to ABA induction, and ACE, BOX4, SP1, GATA which involved in light responsiveness were found. The MBS, GGTCA, TGACG-motif, CGTCA-motif which specifically respond to MeJA were also detected. Another kind of important element TCA-box that involved in defense and stress responsiveness to salicylic acid were found.5. RT and Northern assay were carried out to ascertain the induction profile of GMPase and YIPPEE. Primers were designed according to the complete RNA s or cDNAs. Expression assay showed that the transcripts of two genes started to increased from 6 h after induction, and the level can be detected until 5 d.6. The constructed vectors for promoter characterization were prepared. Candidates of promoter regions were cloned and replaced for the 35S sequence of GUS gene in the pCAMBIA1301 for further study. |
