Cloning and functional analysis of the BrCUC2 gene in Brassica rapa L

Xiaolei Tao; Yuhong Zhao; Li Ma; Junyan Wu; Rui Zeng; JinTang Jiao; Rong Li; Weiming Ma; Yintao Lian; Wangtian Wang; Yuanyuan Pu; Gang Yang; Lijun Liu; Xuecai Li; Wancang Sun

doi:10.3389/fpls.2023.1274567

Cloning and functional analysis of the BrCUC2 gene in Brassica rapa L

Front Plant Sci. 2023 Oct 30:14:1274567. doi: 10.3389/fpls.2023.1274567. eCollection 2023.

Authors

Xiaolei Tao^{1

2}, Yuhong Zhao³, Li Ma^{1

2}, Junyan Wu^{1

2}, Rui Zeng^{1

2}, JinTang Jiao^{1

2}, Rong Li^{1

2}, Weiming Ma^{1

2}, Yintao Lian^{1

2}, Wangtian Wang^{1

2}, Yuanyuan Pu^{1

2}, Gang Yang^{1

2}, Lijun Liu^{1

2}, Xuecai Li^{1

2}, Wancang Sun^{1

2}

Affiliations

¹ State Key Laboratory of Aridland Crop Science, College of Agronomy, Gansu Agricultural University, Lanzhou, China.
² Gansu Key Laboratory of Crop Genetic Improvement and Germplasm Innovation, Gansu Agricultural University, Lanzhou, China.
³ Gansu Yasheng Agricultural Research Institute Co. Ltd, Crop Office, Lanzhou, China.

Abstract

The CUP-SHAPED COTYLEDON2 (CUC2) gene plays an important role in the formation of apical meristem and organ edges in plants. The apical meristematic tissue of Brassica rapa (B. rapa) is associated with cold resistance, however, the role of the CUC2 gene in cold resistance of B.rapa is unclear. In this study, we used bioinformatics software to analyze the structure of BrCUC2 gene, real-time fluorescence quantitative PCR to detect the expression level of BrCUC2, constructed transgenic Arabidopsis thaliana by the flower dipping method and subcellular localization for functional validation. The results showed that, we isolated a 1104 bp open reading frame of BrCUC2 from the winter B. rapa cultivar 'Longyou 7'. The BrCUC2 contains a highly conserved domain belonging to the NAM superfamily. Its homologus CUC genes contain similar conserved motifs and are closely related to Brassica oleracea (B.oleracea), and the N-terminal of amino acid sequence contains NAC domain. BrCUC2 protein was localized in the nucleus and self-activation tests showed that pGBKT7-BrCUC2 had self-activation. Tissue-specific expression analysis and promoter β-Glucuronidase (GUS) activity showed that BrCUC2 had high expression levels in B. rapa growth points and A. thaliana leaf edges, stems and growth points. After low-temperature stress, BrCUC2 showed greater expression in 'Longyou 7,' which presents strong cold resistance and concave growth points, than in 'Longyou 99,' which presents weak cold resistance and protruding growth points. BrCUC2 promoter contains multiple elements related to stress responses. BrCUC2 overexpression revealed that the phenotype did not differ from that of the wild type during the seedling stage but showed weak growth and a dwarf phenotype during the flowering and mature stages. After low-temperature treatment, the physiological indexes and survival rate of BrCUC2-overexpression lines of Arabidopsis thaliana (A. thaliana) were better than those of the wild type within 12 h, although differences were not observed after 24 h. These results showed that BrCUC2 improved the low-temperature tolerance of transgenic A. thaliana within a short time. It can provide a foundation for the study of cold resistance in winter B. rapa.

Keywords: BrCUC2; Brassica rapa; functional analysis; low-temperature stress; transgenic Arabidopsis thaliana.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Natural Science Foundation of China (32260519), Gansu Provincial Technology Innovation Guidance Program (23CXNA0041), Science and Technology Program of Gansu Province (22ZD6NA009), Gansu Province Modern Cold and Arid Agriculture Science and Technology Support (KJZC-2023-12), and the China Agriculture Research System of MOF and MARA (CARS-12).