Objective This paper identifies and explores the role of PtDof family genes under low temperature stress, aiming to explore the non CBF dependent cold signaling pathway of coniferous trees in response to low temperature stress, and provide important basis for the analysis of molecular regulatory mechanism of cold resistance in Pinus tabuliformis and stress resistant molecular breeding.

Method Using transcriptome sequencing to identify differentially expressed genes during the low temperature process in P. tabuliformis, we identified the members of PtDof family genes and conducted bioinformatics analysis, phylogenetic analysis, and expression pattern analysis, screened the key response gene PtDof1 to low temperature stress in PtDof family, and cloned and subcellular localized it. Using agrobacterium mediated inflorescence infiltration method to transform Arabidopsis thaliana, the differences in phenotype and antioxidant enzyme activity between wild-type and overexpressing PtDof1 arabidopsis lines after low temperature treatment were compared and analyzed, and the function of PtDof1 in plant response to low temperature stress was explored.

Result (1)After low temperature treatment, the content of malondialdehyde in needles continued to decrease, while POD activity, SOD activity, and proline content increased. (2) A total of 19 869 differentially expressed genes, including 640 significantly up-regulated genes and 446 significantly down-regulated genes were found in the seedlings of P. tabuliformis at low temperature. (3) A total of 30 Dof genes were identified, and the gene expression patterns in response to low temperature were significantly different, PtDof1 was significantly associated with cold stress. (4) Heterologous transformation showed that the overexpression of PtDof1 improved cold resistance of transgenic Arabidopsis thaliana, and the oxidative stress response was significantly increased.

Conclusion The PtDof1 gene in P. tabuliformis can respond to low temperature stress and may enhance plant cold tolerance by enhancing antioxidant enzyme activity. The research results have demonstrated the existence of non CBF dependent cold signaling pathways in P. tabuliformis, which not only provides a preliminary theoretical basis for analyzing the function of PtDof1 in P. tabuliformis, but also provides important genetic resources for genetic improvement of cold resistant germplasm resources in conifers.