Identification of bZIP gene family of Taraxacum kok-saghyz Rodin and functional analysis of highly expressed genes in latex

【Objective】 This study aimed to explore the role of bZIP gene family of Taraxacum kok-saghyz Rodin in regulating natural rubber biosynthesis， so as to provide foundations for elucidating the regulation mechanism of natural rubber biosynthesis of bZIP transcription factor. 【Method】 Members of the bZIP gene family were identified from the whole genome of Taraxacum kok-saghyz Rodin using bioinformatical methods to predict and analyze their gene structures， conserved motifs， and domains of encoded protein. Based on the data of transcriptome sequencing， candidate genes in latex tissues with high expression and tissue-specific expression were identified. The coding sequences （CDSs） of candidate genes was cloned using PCR， and subcellular localization， transcriptional self-activation activity， and protein interaction analyses were performed. Genetic transformations of the established gene knockout vector pCAMBIA1300-pYAO-cas9-TkbZIPs and overexpressed vector pCAMBIA1300-35S-TkbZIPs-GFP were conducted to identified TkbZIPs gene knockout lines and overexpressed lines. 【Result】 A total of 53 bZIP gene family members were identify from Taraxacum kok-saghyz Rodin. Based on transcriptome data of different tissues， four target genes of TkbZIP1， TkbZIP2， TkbZIP3， and TkbZIP4 were identified， and their CDS lengths were 468， 564， 465， and 1092 bp， encoding 155， 187， 154， and 363 amino acid residues， respectively. Based on real-time fluorescence quantitative PCR， TkbZIP3 and TkbZIP4 genes had high expression in latex， with TkbZIP4 being specifically expressed in latex， and TkbZIP1 and TkbZIP2 genes showed lower expression in latex without obvious tissue-specific expression. Subcellular localization analysis showed four TkbZIPs proteins localized in the nucleus. Yeast transcriptional self-activation assays indicated that TkbZIP1， TkbZIP2， and TkbZIP3 possessed transcriptional self-activation activity， while TkbZIP4 did not have possessed transcriptional self-activation activity. Bimolecular fluorescence complementation （BiFC） assays demonstrated that TkbZIP1， TkbZIP2， TkbZIP3， and TkbZIP4 proteins could form heterodimers through interaction， and the four TkbZIPs proteins could form homodimers through self-interaction. Genetic transformation yielded TkbZIP2 and TkbZIP3 overexpressed lines and TkbZIP1， TkbZIP2， and TkbZIP3 knockout lines. 【Conclusion】 TkbZIP1， TkbZIP2， TkbZIP3， and TkbZIP4 genes exhibit high expressions in latex， suggesting their pivotal regulatory roles in natural rubber biosynthesis. The four TkbZIPs proteins with high expressions in latex could form both homodimers or heterodimers， indicating that bZIP proteins may regulate downstream gene expression in dimeric forms. Gene overexpression materials from TkbZIP2 and TkbZIP3， as well as knockout materials from TkbZIP1， TkbZIP2， and TkbZIP3， were successfully obtained for further investigation of the regulatory role of bZIP gene family in natural rubber biosynthesis.