Molecular Cloning And Transformation Researching Of Phyllotaxis-related Candidate Gene Zmphy In Maize

Maize mutant with opposite phyllotaxy is a good material for DevelopmentalBiology. Excavating and cloning genes associated with it has important theoreticalsignificance and application value which in revealing molecular mechanisms ofdevelopment and conversion of maize phyllotaxy, innovating and riching leafsequence development theory and cultivating new varieties of maize. In this study, thetarget gene Zmphy is derived from candidate genes which is related to the regulationof opposite phyllotaxis out of preliminary analysisd and screened. By cloning andanalysising, overexpressing and RNA interferencing, and maize transforming of thetarget gene, and determining cytokinin and auxin for transformation plants,meanwhile cloning homologous genes in rice named Osphy, main results haveobtained as follows:1. Zmphy was obtained by RT-PCR. The length of this gene is1043bp, encoding164amino acid. The length of ORF frame is492bp.2. Applying bioinformatics methods to study the gene and its encoded amino acidwith a systematic analysis, the results show: Zmphy protein molecular weight is18.19kD, and the isoelectric point is9.08, which is a basic protein.This protein is ahydrophobic protein and and does not have a transmembrane domain. Using theprediction model related sites this experiment also predict the protein secondary andtertiary structure.3. Using RT-PCR to analysis different tissue-specific expression-level of Zmphy.The gene expression in various tissues in descending order: shoot tips, roots, stems,tassel, cornsilk, leaves, shoot tips of which is the highest level of the expression, andthe lowest level of the expression are leaves.4. Using the gene gun, we send the Zmphy into onion epidermal cells. The resultshows that Zmphy mainly expresses in the nucleus.5. Overexpression vectors and interference expression vectors are constructed. weobtained the transformed plants by using in situ transformation method andAgrobacterium-mediated transformation of plant tissue culture respectively, and afterscreening of molecular detection we have obtained positive transgenic plants:4T1generation RNAi transgenic maize of Zmphy;2T0generation overexpressiontransgenic rice of Osphy;4T0generation generation RNAi transgenic rice of Osphyï¼›3T0generation overexpression transgenic rice of Zmphy.6. The study compare the contents of endogenous Cytokinin and auxin betweenwild maize and T1generation RNAi transgenic maize of Zmphy by ELISA. It suggeststhat the contents of Cytokinin are decreased, of which the max is23.85%, while thecontents of auxin is improved, of which the max is49.88%.