Cloning Of Relative Gene About The Regulation And Control Of Florescence Mechanism In The Petal Of Chrysanthemum

Gibberellin(GA), an important plant phytohormone, plays a critical role in the whole lifecycle of higher plant. GA20-oxidase and GA3-oxidase, belonging to multi-gene family, involved in the formation of bioactive GAs, are two key enzymes involved in GA biosynthetic pathway. Regulating the expression of GA20-oxidase and GA3-oxidase will affect the formation of bioactive GAs, which in turn influences the plant growth and development. Taking Chrysanthemum×morifolium'Tangyujinqiu'as experiment material, this study cloned partial fragments of different members of GA20-oxidase genes family and GA3-oxidase gene family, which is helpful for obtaining the whole cDNA and understanding the role of GA in chrysanthemum growth and development. The main results are as follows:1,Designing degenerate primer CM1 and CM2 according to the conserved domain of GA20-oxidase, three partial fragments GA20-oxidase(GA20-ox1,GA20-ox2 and GA20-ox3) were amplificated from Chrysanthemum×morifolium'Tangyujinqiu'. The length of the nucleotides was 486bp which encoded 162 amino acids. The three partial fragments have been submitted to the GenBank databases under the accession number of EU491953, EU491954 and EU491955.2,With the DNAman software, the nucleotides and amino acids sequences of CmGA20-ox1, CmGA20-ox2 and CmGA20-ox3 were analyzed. The homologies aimed to 97.8℅ and 98.8℅ respectively, particularly only one different amino acid between CmGA20-ox1 and CmGA20-ox3. The partial amino acid fragments of CmGA20-ox1 were blasted with the GA20-ox amino acid fragments of tobacco(AB109762), tree peony(ABQ52488), grape(ABF61825) and pea(CAA62846), it was found that the homologies all aimed to 65%. Then GA20-ox phylogenetic trees were constructed according to homologous analytic results, and it indicated the chrysanthemum had the closest genetic relationship with pea.3,Analysis results showed: three cloned GA20-oxidase partial fragments of chrysanthemum all included the conservative sequences-NYYPXCQKP correlated with 2-oxoglutarate combination, conservative H and D residue, and LPWKET motif correlated with GA substrate combination.4,Undertaking conservative domain-search in the amino acid sequences of CmGA20-ox1, CmGA20-ox2 and CmGA20-ox3, the paper found that the three sequences greatly homologized with 2OG-Fe(II) oxygenase superfamily, and the E-value was 2e-23, which contained 86 amino acids from the 77th to the 162nd of the cloned amino acid sequences.5,Designing degenerate primer CM3 and CM4 according to the conserved domain of GA3-oxidase, two partial fragments of GA3-oxidase (GA3-ox1 and GA3-ox2) were amplificated in Chrysanthemum×morifolium'Tangyujinqiu'. The length of nucleotides were 276bp, which encoded 92 amino acids and have been submitted to the GenBank databases under the accession number of EU697529 and EU697530.6,With the DNAman software, the nucleotides and amino acids sequences of CmGA3-ox1 and CmGA3-ox2 were analyzed. The homologies aimed to 94.2℅ and 94.5℅ respectively, and there were five different amino acids between CmGA3-ox1 and CmGA3-ox2. The partial amino acid fragments of CmGA3-ox1 were blasted with the GA3-ox amino acid fragments of tobacco(AB070984), pumpkin(CAC83089) and kidney bean(CAH69705), the homology aimed to 55%. Then GA3-ox phylogenetic trees were constructed according to the homologous analytic results, and it was found that the chrysanthemum has the closest genetic relationship with tobacco.