Functional Differentiation of the Duplicated Gene BrrCIPK9 in Turnip (Brassica rapa var. rapa)

Gene duplication is a key biological process in the evolutionary history of plants and an important driving force for the diversification of genomic and genetic systems. Interactions between the calcium sensor calcineurin B-like protein (CBL) and its target, CBL-interacting protein kinase (CIPK), play important roles in the plant's response to various environmental stresses. As a food crop with important economic and research value, turnip (Brassica rapa var. rapa) has been well adapted to the environment of the Tibetan Plateau and become a traditional crop in the region. The BrrCIPK9 gene in turnip has not been characterized. In this study, two duplicated genes, BrrCIPK9.1 and BrrCIPK9.2, were screened from the turnip genome. Based on the phylogenetic analysis, BrrCIPK9.1 and BrrCIPK9.2 were found located in different sub-branches on the phylogenetic tree. Real-time fluorescence quantitative PCR analyses revealed their differential expression levels between the leaves and roots and in response to various stress treatments. The differences in their interactions with BrrCBLs were also revealed by yeast two-hybrid analyses. The results indicate that BrrCIPK9.1 and BrrCIPK9.2 have undergone Asparagine-alanine-phenylalanine (NAF) site divergence during turnip evolution, which has resulted in functional differences between them. Furthermore, BrrCIPK9.1 responded to high-pH (pH 8.5) stress, while BrrCIPK9.2 retained its ancestral function (low K+), thus providing further evidence of their functional divergence. These functional divergence genes facilitate turnip's good adaptation to the extreme environment of the Tibetan Plateau. In summary, the results of this study reveal the characteristics of the duplicated BrrCIPK9 genes and provide a basis for further functional studies of BrrCBLs-BrrCIPKs in turnip.