Cloning And Characterization Of CgZFP1Gene From Chrysanthemum Grandifolium

Chrysanthemum, Chrysanthemum×morifolium (Compositae, Anthemideae), has historical roots in China, and is one of the ten most famous flowers in China, which is used as the potted flower, cut flower, and applied as the ground-cover plant. Chrysanthemum is valuable for its ornamental character, and is very important for the flower production. With the chrysanthemum cultivation area has increasing, soil salinity has become a major chrysanthemum annual supply constraints. The existence of traditional breeding breeding cycle is long, with no directional characteristics such as lack of improvement, so in recent years, Breeding of genetically modified organisms has targeted biotechnology breeding improved by the advantages of a target character was used in many plant resistance breeding, and excellent resistance gene reserves is the premise of genetic engineering breeding for resistance. Research of CgZFP1with other reported stress-responsive C2H2-type zinc finger proteins, in order to obtain new germplasm resistance in chrysanthemum. The main results are as follows:In this report, a full length cDNA of zinc finger protein gene named CgZFP1was isolated from Chrysanthemum crassum by means of RT-PCR and RACE, the degenerate primers used in this experiment were designed based on the conservative sequences of other stress-responsive C2H2-type zinc finger protein promulgated in GenBank. The full length cDNA of CgZFP was966bp, and the ORF was744bp which was predicted to encode a polypeptide of248amino acid residues. Sequence analysis showed that CgZFPl contained two typical C2H2zinc finger domains. CgZFPl had a similar sequence with ZFP2-3, Mt-ZFP1and CaZFPl, came from Petunia x hybrida, Medicago truncatula and Capsicum annuum and the identities were55.86%,53.57%and54.44%respectively. Phylogenetic tree analysis showed that CgZFP1and other C2H2-type zinc finger protein with two zinc finger domain were grouped into the same branch.Quantitative real-time PCR (qRT-PCR) showed that CgZFP1transcript abundance increased rapidly in response to drought, salinity or cold stress within3h of the stress being imposed. After a24h exposure to200mM NaCl, its expression level gradually increased and reached the peak at12h, thereafter a slight decrease. Its expression peaked within3h of PEG being added to the medium, and then gradually decreased. CgZFP1expression was also induced by low temperature, peaking6h after the beginning of the cold treatment and thereafter declining to a relative stable level. CgZFP1expression was not induced by100μM ABA, the expression level had no obvious difference before and after ABA treatment.The experimental principle to test transcriptional activation ability was shown that, there was a reporter gene His3and an activated sequence UAS in the genome of yeast cell YH109strain. Yeast cells harboring pCL1and pGBKT7-CgZFP1could grow well on SD/-His-Trp, Transcriptional activation of the CgZFPl analysis revealed that CgZFP1possesses transcriptional activation ability in yeast.A plant expression vector for CgZFP1gene pCAMBIA-1301-CgZFP1was constructed in this study, which was introduced into Arabidopsis thaliana via an Agrobacterium tumefaciens EHA105-mediated floral dip method. Two independent transgenic lines overexpressing CgZFP1were selected for further investigations and compared with wild-type A. thaliana. The expression of CgZFP1was increased from the transgenic lines35S::CgZFP1-2and35S::CgZFPl-12compared with A. thaliana wild-type plants by quantitative real-time PCR. The stress assays showed that the expression level of CgZFPl in A. thaliana significantly increased. The expression level of a number of genes associated with the stress response in plants including Atlea3, AtP5CS2, AtProTl, AtMnSOD, AtPOD, AtAPXl,AtSOSl,AtSOS2, AtSOS3,AtNHX1, AtPIP2A, AtMYB21was raised in the CgZFPl transgenic Arabidopsis plants.The results indicate that, under the drought and high salt stress, CgZFP1may control osmotic adjustment, antioxidant levels; and high salt stress, the gene can also start the ion transport, water stress can regulate water transport genes.