Abstract: [Objective] The expression patterns of 6 cold resistance related genes (RAP2.7, ABR1, AtHSFA6B, AtbZIP44, GRXC6 and HSP18.2) in cold-tolerant and cold-intolerant Casuarina equisetifolia were analyzed in order to provide a theoretical basis for elucidating the molecular mechanism of Casuarina trees in response to cold stress.[Method] The relative conductivity in cold-tolerant (ZS7) and cold-intolerant (HN1) clones of C. equisetifolia were measured under low temperature stresses at -2——11℃. The low half-lethal temperature (LT₅₀) were calculated by fitting the temperature with the relative conductivity based on the logistic equation. Quantitative Real-Time PCR (qPCR) was employed to investigate the differential expression of the 6 cold resistance related genes in cold-tolerant and cold-intolerant clones under successive low temperature stresses at -2, -5 and -8℃, as well as precise expression patterns under low temperature stresses at -5℃ for 1, 2, 5, 8, 16, 24, 48 and 72 h. The specific primers for qPCR were designed based on the EST sequences of 6 cold resistance related genes identified from previous transcriptome analysis. Analysis of relative gene expression data was performed using 2^-ΔΔC_Tmethod.[Result] Under low temperature stresses, relative conductivity in cold-tolerant and cold-intolerant clones had a very highly significant difference (P<0.01), LT₅₀ was -5.92℃ and -2.87℃, respectively. Under normal temperature condition, the relative expression of all the 6 genes in 2 clones had no significant differences. However, under low temperature stress at -2℃ for 2 h, which was close to the LT₅₀ of cold-intolerant clone, expression of these genes in the cold-intolerant clone were strongly inhibited, while in the cold-tolerant clone were activated. Under low temperature stress at -5℃ for 2 h, which was close to the LT₅₀ of the cold-tolerant clone, expression of these genes were further inhibited in the cold-intolerant clone, also in the cold-tolerant clone. Under low temperature stress at -8℃ for 2 h, which was lower than the LT₅₀, the expression of all 6 genes in 2 clones were inhibited continuously. The precise expression patterns analysis revealed that the expression of all 6 genes were significantly up-regulated in the cold-tolerant clone under low temperature stress at -5℃ for 8 h, reaching the maximum at 8 h, 24 h and 48 h, and significantly down-regulated in the cold-intolerant clone at 1-16 h, reaching the minimum at 1-5 h.[Conclusion] On the expression level of cold resistance related genes, different clones showed significantly different responding mechanisms to low temperature stress. Low temperature induces the expression of genes belonging to transcription factors and ROS family, and genes related with the response to ROS in cold-tolerant C. equisetifolia to resist or adapt to cold stress, instead it was inhibited in the cold-intolerant C. equisetifolia, thus significantly decreased its adaptation to cold stress. This study provided a useful basis for enriching the molecular mechanism of Casuarina trees in coping with cold stress, and for molecular selection and breeding of cold-tolerant Casuarina clones.

Key words: Casuarina equisetifolia, low temperature stress, cold tolerance, gene expression, molecular breeding