Cloning, Characterization, and Functional Analysis of EuTIL1, a Gene-Encoding Temperature-Induced Lipocalin in Eucommia ulmoides Oliv

Abstract

Eucommia ulmoides Oliver is a unique tertiary relict tree species in China belonging to the Eucommia family and genus. It is a traditional and precious Chinese medicinal herb with anti-tumor, antibacterial, antioxidant, anti-inflammatory, and bidirectional blood pressure regulation effects. Eucommia ulmoides mainly grows in temperate regions of China, but due to its sensitivity to low-temperatures, it is difficult to introduce into new regions. To study the role of Eucommia ulmoides lipocalin in plants. This investigation was conducted utilizing gene cloning, bioinformatics analysis, quantitative real-time polymerase chain reaction (qRT-PCR), subcellular localization, and stable genetic transformation to transfer EuTIL1 into Nicotiana tabacum Xanthi. The wild-type transgenic vector and EuTIL1 tobacco were cold-treated, and the corresponding protective enzyme activity and cold-induced gene expression levels were measured to analyze the functions of the genes. In this study, the full-length of the temperature-induced lipocalin gene (EuTIL1) cDNA was cloned from the leaves of Eucommia ulmoides using the rapid amplification of cDNA ends (RACE) method. The sequence analysis showed that the full-length cDNA of EuTIL1 was 917 bp and encodes a protein of 188 aa residues, which is a member of the Lipocalin-2 family. Subcellular localization analysis revealed that EuTIL1 was found in the plasma membrane. The transgenic tobacco lines expressing EuTIL1 under the control of the CaMV 35S promoter had increased tolerance to cold compared to wild-type (WT) plants. The average water loss rate of EuTIL1 transgenic plants was 12.4%, the average conductivity at 24 h was 55.11%, and the malondialdehyde content at 24 h was significantly lower than that of wild-type plants. The maximum soluble sugar (SS) content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, and catalase activity of EuTIL1 plants after low-temperature treatment were 22.03 mg/g, 726.87 U/g, 1283.94 U/g, and 356.84 U/g, respectively, which are significantly higher than those of the wild-type. Meanwhile, in the EuTIL1 transgenic tobacco plants, the expression of the NtDREB1, NtDREB2, NtDREB4, and NtCOR15a elevated under the low-temperature treatment condition. In conclusion, our study demonstrates that EuTIL1 is a gene involved in the cold-stress response and has the potential to enhance cold tolerance in plants, providing a potential molecular basis for the study of Eucommia ulmoides introduction and serving as a candidate gene for evaluating cold-tolerant plants.