| CPPU is a synthetic cytokinin-active phenylurea with a physiologic activity exceedingthat of zeatin. Preliminary experiments found that CPPU could induce wheat tillering.Tillering is one of the important agronomic traits that affect the panicles and yield of majorcrops such as wheat. To identify differential proteins induced by CPPU, two-dimensionalelectrophoresis and mass spectrometry were used.Among the differential proteins, two proteins were closely related to auxin and cytokininsynthesis and maybe related to tillering. Two novel genes of TaYUCCA and TatRNA IPT werecloned from wheat and their expression characteristics were analyzed. The function ofTaYUCCA was studied initially. The main results are as follows:1. The effects of CPPU on tillering of wheat. The research proved that CPPU could inducewheat tillering, and the approciate concentratin which induces the largest number of tillerswas6μmol/L. The results of fields showed that with increasing concentration of CPPUtreatment the number of effective tillers of wheat increased and then showed a downwardtrend, and wheat have the largest number of tillers when under6μmol/L CPPU treatment.2. Analysis of differential proteins induced by Forchlorfenuron in wheat. Compared withthe control, the expression of10protein spots was up-regulated. These protein spots werechosen for further analysis. The CPPU-responsive proteins were excised from preparative gelsand examined via MALDI-TOF/TOF MS analysis using MALDI-TOF/TOF. The10proteinsidentified were tRNA delta(2)-isopentenylpyrophosphate transferase, a probable peptide ABCtransporter ATP-binding protein y4tS, ribosomal RNA large subunit methyltransferase N,microtubule-associated protein6, actin-66(Fragment), mitochondrial ATP synthase subunitalpha, S-adenosylmethionine synthase1, flavin-containing monooxygenase YUCCA4,GTPase obg, and ribosomal RNA small subunit methyltransferase G. These proteins wereinvolved in phytohormone synthesis, plant stress response, energy metabolism, proteinsynthesis, cytoskeleton constructing, and transportation.3. Two novel genes, TatRNA IPT and TaYUCCA, were cloned from wheat and their sequences were analysed. The open reading frames (ORF) of TatRNA IPT and TaYUCCAencoded a466-polypeptide and a382-polypeptide respectively. The deduced sequence ofTatRNA IPT and TaYUCCA had no signal peptide, and therefore, they may carry out functionsin cytoplasm. As shown in the alignment, the predicted sequences of TatRNA IPT had aconserved domain for P-loop NTPase superfamily, and the predicted sequences of TaYUCCAhad a conserved domain for Pyr redox superfamily. The phylogenetic tree revealed that theputative polypeptide of TatRNA IPT had significant homology with OstRNA IPT, andTaYUCCA had high homology with BdYUCCA.4. The expression analysis of TaYUCCA. The real-time quantitative reverse transcriptionPCR showed that, compared with the control, TaYUCCA was up-regulated after treated with6μmol/L CPPU for24and48hours. Use the four parts of germinating wheat embryo forRT-PCR analysis, the results showed that TaYUCCA was more expressed in tillering parts androots than the top and middle of bud.5. Plant expression vector for TaYUCCA was constructed successfully. TransgenicTaYUCCA Arabidopsis was obtained by Agribacterium tumefaciens-mediated transformation.Compared with the Wild-type Arabidopsis, transgenic TaYUCCA Arabidopsis germinated later,the hypocotyl was obviously elongation and the leaves were curl.In conclusion, we identified the differential proteins induced by CPPU usingtwo-dimensional electrophoresis and mass spectrometry. TatRNA IPT and TaYUCCA werecloned from wheat. They related to auxin and cytokinin synthesis. their sequencecharacteristics and expression patterns were analyzed. Plant expression vectors of TaYUCCAwere constructed and the function of TaYUCCA was analyzed. These genes maybe related towheat tillering. The above works established a foundation for studying the machenisim ofwheat tillering. |
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