Identification and Expression Analysis of CKX Gene Family in Brassica juncea var. tumida and Their Functional Analysis in Stem Development

Cytokinin oxidase/dehydrogenase (CKX) is a specific enzyme affecting plant growth, development, and yield by mediating the metabolism of endogenous cytokinins in a dual catalytic mode. This study aims to reveal the distribution and associated properties of CKX gene family members in the whole genome of Brassica juncea, lay a theoretical basis for further exploration of the biological function of BjuCKX genes, and provide genetic resources to improve the breeding quality of B. juncea. We screened BjuCKX genes with typical FAD-binding and CK binding domains and identified them using bioinformatics methods. In addition, we analyzed physicochemical properties, phylogenetic relationship, gene structure, conservative motif, cis-acting element of promoter, and expression pattern of BjuCKX gene family members. Endogenous hormone levels (GA3, ZR, IAA, ABA, BR and MeJA) were also determined in different developmental periods using an indirect enzyme-linked immunosorbent assay (ELISA). A total of 23 BjuCKX genes were identified, and they were renamed BjuCKX01-BjuCKX23. Further analysis revealed that the amino acid number of 23 BjuCKX proteins ranged from 333 to 1337 aa, the molecular weight ranged 36.58 to 148.49 kDa, whereas the theoretical isoelectric point ranged from 4.94 to 9.10. The phylogenetic tree clustering analysis can group family members into four subgroups. Collinearity analysis revealed that genes were not evenly distributed on the chromosome, with a pair of tandem repeats. Meanwhile, BjuCKX genes located on each chromosome revealed cross collinearity caused by fragment replication. The genes were more conserved in structure. In the upstream region of promoter, there were several cis-acting elements, including plant growth and development, hormone response, and biological and abiotic stress. Combined with transcriptome data, BjuCKX gene expression has been demonstrated to be different at varying developmental stages of the stem. RT-qPCR further confirmed that BjuCKX genes were involved in stem development and affects growth by regulating endogenous hormone levels.