Cloning And Functional Identification Of Transcription Factor CpNAC68 Gene In Wintersweet [Chimonanthus Praecox(L.)Link]

Wintersweet[Chimonanthus praecox（L.）Link]is a deciduous shrub or small tree of the genus Chimonanthus in the Chimonanthus family,which is an endemic specie in our country.Because it flowering in cold winter with unique fragrance and beautiful flower shape,it is widely used in bonsai production,cut flower production,road greening,biopharmaceutical and so on.Wintersweet has strong abiotic stress tolerance.However,the research on the molecular mechanism of stress resistance is still relatively limited.NAC（NAM,ATAFs,CUC）,as one of the largest family of transcription factors unique to plants,which have important regulatory roles in the entire growth and development process of plants and in response to biotic and abiotic stress.Currently,the investigation of molecular mechanism is mainly focused on model plants and crops.In order to enrich the understanding of the biological functions of the NAC gene family of wintersweet.In this study,CpNAC68 gene was cloned.The basic characteristics such as its molecular characteristics,subcellular localization,transcriptional activity and expression in wintersweet seedlings were inllustrated.The abiotic stress tolerance of overexpressed plants was analyzed by overexpression in Arabidopsis thaliana.We finally provided a newly theoretical basis to further analyze the resistance molecular mechanism of wintersweet.The specific research results are as follows:1.Cloning and molecular propoties of CpNAC68Based on the intrinsic transcriptome database of the wintersweet in Chongqing Engineering Research Center for Floriculture.A cDNA sequence with a complete open reading frame（ORF）was cloned by PCR technology,named CpNAC68.The length of full sequence of CpNAC68 cDNA is 1336 bp,including the largest ORF of 906 bp,encoding for 302 amino acid.Through homologous comparison,we found that CpNAC68protein has a high similarity with NAC68 protein of other plants.The N-terminal had a conserved domain,including A,B,C,D and E subdomains,while the C-terminal is highly variable.According to the online prediction software,CpNAC68 is a stable hydrophilic protein without signal peptide and transmenmbrane domain.CpNAC68 belongs to the NAM subfamily and has a close relationship with Nelumbo nucifera.2.Subcellular localization analysis and transcriptional activation assay of CpNAC68（1）The subcellular localization vector p CAMBIA300-CpNAC68-GFP was constructed and transformed into Agrobacterium,then infected tobacco epidermal cells,CpNAC68-GFP was only distributed in the nucleus under fluorescence confocal microscope.The results indicated that CpNAC68 localized in the nucleus.The yeast expression vector p GBKT7-CpNAC68-ORF was constructed,p GBKT7 was used as a negative control and the expression vector p GBKT7-VP16 was used as a positive control.Transferred the three vectors into the yeast Y2H-Gold.The experimental results elucidated that CpNAC68 has a transcriptional activation function.3.Expression analysis of CpNAC68Quantitative Real-time PCR was used for analysis the expression pattern of CpNAC68 in different tissues,flowering stages,organs of full bloom,abiotic stress including high temperature（42℃）,low temperature（4℃）,high salt（300mmol Na Cl）,high osmotic pressure（PEG osmotic stress）and hormone treatment（JA,GA,SA）of wintersweet seedlings.The experimental results show that CpNAC68 expressed different degrees in different tissues,flowering stages,floral organs,among which CpNAC68 is highest expressed in old leaves.The expression level in full bloom and decline were the highest.Moreover,CpNAC68 is highest expressed in pistil compare with other floral organs.It is speculated that this gene is involved in the senescence progress of leaves and flowers,as well as the development of pistil.When it comes to abiotic stress and hormone treatment,CpNAC68 showed a up-regulated expression trend under high temperature,low temperature,high salt,JA,and GA treatments,while down-regulated expression trend under high osmotic pressure and SA treatment.Therefore,CpNAC68 may complicatedly regulated by various environments and hormones.4.Phenotypic observation and abiotic stress research of transgenic A.thalianaAn overexpression vector p GWB551-CpNAC68 was constructed and transferred into A.thaliana by Agrobacterium-mediated inflorescence infection.Transgenic lines was obtained by hygromycin（Hyg）screening.Quantitative Real-time PCR technology was used to determine the high,middle and low expression level of homozygous T₃ generation in order to performed phenotypic observation and abiotic stress treatment.The results elucidated that the transfer of CpNAC68 into the col-0 type A.thaliana will not affect the growth and development of transgenic plant.Moreover,under drought,high osmotic pressure,high temperature,high salt,transgenic type have higher survival rate and chlorophyll SPAD value than wild type.Besides,the relative electrolytic leakage and malondialdehyde molality are lower than that in wild type,transgenic plants exhibit stronger overall stress resistance than wild type.We can conclude that CpNAC68participates in the process of plants responding to multiple stresses and enhances the stress resistance of transgenic A.thaliana.