Bioinformatics Of WD40 Gene Family In Safflower And The Cloning And Preliminary Characterization Of CtWD40-1

The WD40 transcription factors belong to a superfamily of genesthat widely distributed in eukaryotes.The potential role of this super gene family is more likely related to plant growth and development and their adaptation to multiple environments.Recent studies indicated the WD40 transcription factor is specifically associated with biosynthesis of anthocyanins.Anthocyanin is an extensible class of flavonoids found abundantly in safflower,which plays an important role in the treatment of various cardiovascular and cerebrovascular diseases.At present,it has been widely used in the fields of medicine and food,however,the explicit regulatory mechanism of WD40 is still unknown.In this study,40 putative CtWD40 family members located in the safflower genome were identified through bioinformatics analysis including subcellular localization prediction,promoter cis-acting elements,and protein interaction analysis.In addition,the differential expression pattern of 40 members of the CtWD40 subfamily in different tissues and flowering stages of safflower was studied by using qRT-PCR technology.For further functional validation,the WD40 family member gene CCG020780.1(CtWD40-1)was cloned and transiently coexpressed with GFP protein using the onion epidermal cells to experimentally investigate its subcellular localization.Furthermore,to characterize its possible function,we over-expressed CtWD40-1 in Arabidopsis through agrobacterium mediated transformation using floral dip method.The specific research results are as follows:The physics chemical properties suggested the CtWD40 encoding protein molecular weights ranges from 35.296 kDa to 170.134 kDa,and their isoelectric point(PI)ranges from 4.92 to 9.80.The aliphatic coefficient ranges from 51.16 to 93.58.According to GRAVY,CtWD40 protein showed hydrophilic protein nature.According to the prediction of subcellular localization,31 CtWD40 proteins were located in the nucleus,4 CtWD40 proteins were located in the cytoplasm,and the other 5 CtWD40 proteins were located in the chloroplast.Further identification was performed through cis-acting element search of the gene promoters,suggesting the presence of CtWD40 specific promoter elements indicating their possible role in the me Tableolic pathways.Functional enrichment analysis of the CtWD40 protein were mostly enriched in the category of biological functions.During the conserved protein motifs were retrieved from all CtWD40 family proteins,out of which 6 were identified as WD40 specific conserved regions.The results of protein interactions network between CtWD40 proteins,their potential regulatory functions were predicted.The qRT-PCR technology was used to study the differential expression of 40 members of the safflower CtWD40 subfamily in different tissues and flowering stages of safflower.The results showed that all CtWD40 genes were highly related to the process of safflower growth and development,and partly involves in flavonoid synthesis.The WD40 family member gene CCG20708.1 was isolated and effectively expresses transiently with GFP proteins(PCAMBIA1032-CtWD40-1)for subcellular localization.The results showed that CtWD40-1 was located in the nucleus with the help of RT-PCR technology.Finally,to validate gene function of CtWD40-1,we successfully overexpressed it in the Colombian Arabidopsis thaliana using the plant overexpression vector(pCAMBIA3301-CtWD40-1)and T2 transgenic Arabidopsis was obtained.