Pak choi (Brassica rapa ssp. chinensis Makino) is a representative seed vernalization vegetable and premature bolting in spring can cause significant economic loss. Thus, it is critical to elucidate the mechanism of molecular regulation of vernalization and floral bud initiation to prevent premature bolting. Gibberellin (GA) is the key plant hormone involved in regulating plant development. To gain a better understanding of GA metabolism in pak choi, the content of GA in pak choi was measured at different stages of plant development using enzyme-linked immunosorbent assay. The results showed that the GA content increased significantly after low-temperature treatment (4°C) and then decreased rapidly with vegetative growth. During floral bud initiation, the GA content increased rapidly until it peaked upon floral bud differentiation. To elucidate these changes in GA content, the expression of homologous genes encoding enzymes directly involved in GA metabolism were analyzed. The results showed that the changes in the expression of four genes involved in GA synthesis (Bra035120 encoding ent-kaurene synthase, Bra009868 encoding ent-kaurene oxidase, Bra015394 encoding ent-kaurenoic acid oxidase, and Bra013890 encoding GA20-oxidase) were correlated with the changes in GA content. In addition, by comparing the expression of genes involved in GA metabolism at different growth stages, seven differentially expressed genes (Bra005596, Bra009285, Bra022565, Bra008362, Bra033324, Bra010802, and Bra030500) were identified. The differential expression of these genes were directly correlated with changes in GA content, suggesting that these genes were directly related to vernalization, floral bud initiation and development. These results contribute to the understanding of the molecular mechanism of changes in GA content during different developmental phases in pak choi.

Flowering is very important for leaf head formation and breeding. Investigating the key genes related to vernalization process can reveal the flowering regulation mechanism and guide production and germplasm breeding. In this study, two Chinese cabbage (Brassica rapa subsp. pekinensis) inbred lines ‘1#’ and ‘2#’ with different winterness were used to identify vernalization-related genes. After low-temperature treatment, the transcriptomes of the shoot apex of the two lines were analyzed. The Arabidopsis thaliana homologues of 2,932 common differentially expressed genes with the same change trend after vernalization of two inbred lines were analysed based on Gene Ontology annotation and description. The results showed that, three genes were identified that may be related to the vernalization: Bra031210, Bra009126 and Bra033615. In which Bra031210 and Bra009126 were cloned, named BrGRP7.1 and BrCPD1, with open reading frames lengths of 507 and 1443 bp, encoding 168 and 480 amino acids, respectively. Through the expression patterns analysis, the expression of BrGRP7.1 showed a trend of initial decreasing (dipping at stage 1) and then increasing (peaking at stage 5) during flower bud differentiation of both inbred lines. Incontrast, BrCPD1 showed a first decreasing, then increasing, and last decreasing trend during flower bud differentiation of both lines. These two genes were transferred into wild-type A. thaliana. The flowering time of T2 generation of BrGRP7.1- and BrCPD1-transgenic plants was 2.31~5.11 days and 2.64~4.35 days earlier than the wild-type, respectively. In conclusion, this study screened three genes that involved in the vernalization process. Through functional verification, BrGRP7.1 and BrCPD1 were proved to have the ability to promote flowering time. This study provided a reference for further study on the flowering mechanism of Chinese cabbage.