Abstract:
【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.