Abstract:

Objective: This study aims to establish a transient gene silencing system in leaves, in order to elucidate the role of PHYTOENE DESATURASE (PDS) gene of Catalpa bungei, CbuPDS-5, in the carotenoid synthesis pathway of the leaves. Method: In this study, C. bungei was used as the research subject, and the PDS gene family members were identified in the C. bungei genome. The expression pattern analysis was conducted to determine the expression levels of CbuPDSs in various tissues of C. bungei. The homologous cloning method was used to obtain the cDNA sequence of CbuPDS-5 from C. bungei. The structure and classification of CbuPDS-5 were analyzed by constructing a phylogenetic tree and performing homologous protein sequence alignments. The homologous recombination technique was used to construct a TRV2-CbuPDS-5 expression vector, and the Agrobacterium-mediated injection method was used to infect the leaves of C. bungei to create gene-silenced lines. Meanwhile, phenotypic observation and real-time PCR were performed to analyze the effects of CbuPDS-5 silencing on C. bungei growth and leaf pigment synthesis. Result: Six PDS members were identified in the C. bungei genome, with significant differences in their expression levels in different tissues such as leaves and flowers. Among them, the CbuPDS-5, which exhibited the highest similarity to tomato CaPDS, was highly expressed in leaves, and selected as the target gene. The full-length CbuPDS-5 was 1 095 bp, with a 948 bp ORF encoding 315 amino acids. It contained a dinucleotide binding motif, however had an amino acid deletion in the C-terminal. The functional validation showed that new leaves exhibited distinct photobleaching symptoms in two months after inoculation, and repeated inoculations significantly increased the extent of leaf whitening. The qRT-PCR analysis confirmed a significant reduction in the relative expression level of CbuPDS-5 in leaves with exhibiting photobleaching symptoms. Conclusion: The experimental results have demonstrated that the VIGS system can be used for gene silencing in C. bungei. CbuPDS-5 can serve as an indicator gene for rapid verification of the efficiency and duration of transient systems in woody plants. Additionally, the creation of variegated C. bungei through CbuPDS-5 silencing enhances its ornamental value and provides a foundation for breeding dual-purpose varieties with both ornamental and timber uses.

Key words: Catalpa bungei, PHYTOENE DESATURASE (PDS), gene silencing, whitening leaves