Cloning Of The Transcription Factor CpBBX19 Gene And The Function In The Regulation Of Salt And Drought Tolerance

Wintersweet（Chimonanthus praecox）is a deciduous or semi-deciduous small tree or shrub of the Chimonanthus Lindl.of the Calycanthaecae family,and it is also a unique traditional ornamental flower and tree in China.The industry of wintersweet has begun to flourish,and it is often used as bonsai,garden greening and cut flowers,etc.It has a very high ornamental value,as well as a huge market potential and economic value.Wintersweet has a wide distribution range,developed root system and strong resistance to abiotic stress.The BBX gene family is a type of zinc finger protein transcription factor,which is involved in the regulation of multiple growth and development processes of plants,such as photomorphogenesis,shade avoidance,flowering regulation,biotic and abiotic stress.The cloning and functional analysis of the BBX gene of wintersweet will help to provide a new theoretical basis for the transcriptional regulation mechanism of wintersweet stress resistance.In this paper,the transcription factor of Cp BBX19 was explored and cloned its full-length fragment,and the expression characteristics of the gene were analyzed.By heterologous transformation of Arabidopsis,the function of Cp BBX19 in abiotic stress was studied.The main findings are as follows:1.Cloning and molecular characteristics of Cp BBX19On the basis of the sequence obtained from the transcriptome database of the developmental transcriptome of wintersweet,specific primers were designed and PCR was used to amplify the full-length sequence of Cp BBX19 with a complete Opening Read Frame（ORF）.This sequence contained a 5’-UTR（5’-untranslated region）of475bp,a 3’-UTR（3’-untranslated region）of 428 bp and a CDS（Coding Sequence）of633bp,which encodes 210 amino acids.By constructing a phylogenetic tree of Cp BBX19,and comparing the protein encoded by Cp BBX19 with the BBX protein of other species,it is shown that Cp BBX19 belongs to theⅣsubfamily and was similar to the zinc finger protein BBX19 of Ziziphus jujuba（XP＿015892301.1）.Through online analysis,it is speculated that the protein is an unstable hydrophilic protein,and its molecular formula is predicted to be C₉₉₂H₁₅₉₀N₃₀₄O₃₁₆S₂₀.It has no signal peptide structure and no transmembrane domain.The secondary structure of Cp BBX19 protein is mainly composed of random coils（57.14%）,alpha helix（22.38%）,and a small amount of extended strand（17.62%）.2.Expression pattern analysis of Cp BBX19 in wintersweetQuantitative real-time PCR（q RT-PCR）was used to analyze the expression pattern of Cp BBX19 in different tissues,different periods,and different stress treatments（drought,high salt,high temperature,low temperature,ABA,Me JA）in wintersweet.It shown that Cp BBX19 was expressed in all tissues of wintersweet,but the highest expression was in the inner petals,cotyledons and young leaves.It is speculated that this gene may be involved in the morphogenesis of leaves and the development of inner petals.Cp BBX19 was also expressed in all flower development stages,with the highest expression detected in early initiating bloom,followed by late initiating bloom and bloom.The expression of Cp BBX19 was induced by different abiotic stress（cold,heat,Na Cl,and drought）and hormone（ABA and Me JA）treatments,but under different conditions,the time points for the highest expression level were different.The results speculated that Cp BBX19 may respond to drought,high salt,high temperature,low temperature and other stresses,and may be involved in the signal transduction pathway of ABA and Me JA.3.Stress tolerance analysis overexpression of Cp BBX19 gene in ArabidopsisThe plant overexpression vector p GWB-551-Cp BBX19 was constructed,and Arabidopsis transgenic lines（OE lines）were obtained by transferring the recombinant plasmid into wild-type Arabidopsis via the floral dip method.The homozygous T3generation transgenic Arabidopsis was obtained by multiple screening.Three T3transgenic lines（with high,medium,and low expression of Cp BBX19,respectively）were selected for phenotypic investigation.Phenotypic analysis showed that heterologous expression of Cp BBX19 in Arabidopsis enhanced the tolerance of the plants to salt and drought stress.Further studies found that electrolyte leakage and malondialdehyde（MDA）concentrations in transgenic Arabidopsis after stress treatments were significantly lower than those in wild-type（WT）plants.Meanwhile,under salt and drought stress conditions,the expression of At COR15a,At KIN1,At LEA76 and At RD29b genes which were stress-ralated were significantly up-regulated.Furthermore,three stress-related genes（At ABF1,At RAB18 and At RD29a）were significantly up-regulated in the transgenic lines when the seedlings were under normal conditions or under salt and drought stress conditions.The above results indicate that Cp BBX19 can activate the expression of stress resistance-related key genes under the stimulation of abiotic stress,and then participate in the regulation of plant tolerance to abiotic stress.