Cloning And Functional Preliminary Identification Of CpDi19-5 Gene From Chimonanthus Praecox (L.) Link

Chimonanthus praecox（L.）Link is a traditional Chinese famous winter flower.It has a long history of cultivation and high ornamental value,and is widely used in landscape.As a typical winter flowering plant,Chimonanthus praecox has tenacious vitality and has strong cold and drought resistance.It is speculated that it may contain specific stress resistance genes.Previous studies have shown that transcription factors can play a regulatory role in plants responding to different stresses,and are extremely important molecular switches in the stress-resistance signaling pathway.Di19（Drought-induced 19）transcription factor is an important transcription factor closely related to plant response to abiotic stress.Studies in different species have proved that it can be widely involved in regulating plant responses to various stresses such as drought,high salt,and high temperature.At present,the research of Di19 gene is mainly concentrated in model plants,and there is no report on the cloning and functions of Di19 family genes in Chimonanthus praecox.In this study,a Di19 family gene was cloned from Chimonanthus praecox,named CpDi19-5,and its molecular characteristics and functions were preliminary explored.The main results obtained by the experiment are as follows:1.Cloning and molecular characteristics analysis of CpDi19-5 geneA full-length cDNA sequence of the CpDi19-5 gene with a complete ORF frame was cloned by PCR amplification technology using the cDNA library of Chimonanthus praecox.Sequence analysis revealed that the cDNA of the gene was 788 bp in length,contained a maximum open reading frame of 657 bp,and encoded 218 amino acids.The CpDi19-5 protein contains highly conserved zf-Di19 and Di19＿C domains,and is a typical Di19 family transcription factor.The CpDi19-5 protein does not have a transmembrane structure and a signal peptide sequence.It belongs to a hydrophilic protein and is mainly composed of an alpha-helix and an irregular coil.The results of protein sequence homology alignment showed that CpDi19-5 protein had high similarity with Di19 protein in other species.Construction of the evolutionary tree analysis revealed that CpDi19-5 protein was closest to lotus protein Nu Di19-5-like,so it was named CpDi19-5.2.Subcellular localization of CpDi19-5The fusion expression vector pCAMBIA1300-CpDi19-5-GFP was constructed for transient expression in tobacco epidermal cells.It was found that the fusion expression vector had green fluorescence on the nucleus and cell membrane.The results indicate that CpDi19-5 protein is localized on the nucleus and cell membrane.3.Expression pattern analysis of CpDi19-5 gene in Chimonanthus praecoxThe expression pattern of CpDi19-5 gene under different tissues,different flower developmental stages,and different stress and hormone treatment conditions was analyzed using real-time quantitative PCR technology.The results showed that the CpDi19-5 gene was expressed in all tissues of Chimonanthus praecox,but the expression level was higher in various organs of flowers.During the development of Chimonanthus praecox,the expression of CpDi19-5 gene was higher from the open petal stage to the decay stage.The expression level of CpDi19-5 gene was significantly decreased after treatment with PEG and JA;the expression level was significantly increased after treatment with high temperature stress;while the expression level was not significantly changed under Na Cl,low temperature and GA treatment.4.Phenotypic analysis and drought stress study of CpDi19-5 Transgenic Arabidopsis thalianaThe Gateway technology was used to construct the plant overexpression vector p GWB551-CpDi19-5,which was transformed into Arabidopsis by inflorescence infectionmethod.After screening,homozygous T₃ transgenic Arabidopsis were obtained.Three lines with high,medium and low expression levels were selected for phenotypic observation and drought stress experiments.The experimental results show that compared with the wild type,the CpDi19-5 gene does not affect the growth and development of transgenic Arabidopsis;under drought stress,the CpDi19-5 gene significantly reduces the drought stress tolerance of transgenic Arabidopsis.The determination of related physiologyical indicators was also consistent with the phenotype under drought stress,indicating that the CpDi19-5 gene negatively regulates drought tolerance in Arabidopsis.In a word,it was showed that the CpDi19-5 gene of Chimonanthus praecox may participate in the response to drought stress,and can affect the drought stress tolerance in transgenic Arabidopsis.