【摘要】 近年来，钩吻（Gelsemium elegans）的药用和饲用价值日益凸显，但钩吻在生长过程中不耐低温，挖掘其低温响应基因，为钩吻的抗寒育种研究奠定基础。植物中，脂氧合酶（lipoxygenase, LOX）在种子老化、抗逆境胁迫等方面的生理生化过程中有重要影响。基于课题组构建的钩吻转录组数据库，挖掘响应低温胁迫的钩吻LOX基因，运用RT-PCR技术，从中克隆到一条GeLOX1的cNDA全长序列，对其进行生物信息学、亚细胞定位、基因表达、原核表达及平板胁迫等分析。结果显示，GeLOX1所编码蛋白的氨基酸长度为761 aa，蛋白相对分子质量为87.00 kD，预测为不稳定的亲水性蛋白，含有28个丝氨酸磷酸化位点，22个苏氨酸磷酸化位点和9个酪氨酸磷酸化位点。进化树分析结果表明，GeLOX1属于9-LOX家族的成员。亚细胞定位检测结果显示，GeLOX1蛋白定位于细胞质中。实时荧光定量PCR分析发现，GeLOX1在钩吻的根中高表达，且其在4℃低温胁迫下的表达量呈现下调的趋势。经原核表达诱导后，GeLOX1的重组蛋白在约111 kD处出现目标条带，且重组蛋白的积累量在诱导8 h时达到峰值。此外，平板胁迫试验表明，GeLOX1的原核表达菌株相较于对照组对低温胁迫更敏感。钩吻GeLOX1能够应答低温胁迫。更多还原

【Abstract】 In recent years, the medicinal and feeding value of Gelsemium elegans has become increasingly prominent. However, G. elegans is not tolerant to low temperatures during its growth process. Exploring its low temperature response genes may lay the foundation for the research of cold resistance breeding of G. elegans. Lipoxygenase(LOX)has a significant impact on the physiological and biochemical processes of plants in seed aging, stress resistance, and other aspects. The LOX genes of G. elegans in response to low temperature stress were mined from the transcriptome database of G. elegans constructed by our team. A full-length c DNA sequence of the GeLOX1 gene was cloned from the leaves of G.elegans using RT-PCR technology, and its bioinformatics, subcellular localization, gene expression, prokaryotic expression, and plate stress were analyzed. The results showed that the amino acid length of the protein encoded by the GeLOX1 was 761 aa, and the relative molecular weight of the protein was 87.00 kD. It was predicted to be an unstable hydrophilic protein, containing 28 serine phosphorylation sites, 22 threonine phosphorylation sites, and 9 tyrosine phosphorylation sites. The analysis results of evolutionary tree indicated that Ge LOX1 belonged to the 9-LOX family. The subcellular localization test results showed that the GeLOX1 was located in the cytoplasm. Real time fluorescence quantitative PCR analysis revealed that the GeLOX1 was highly expressed in the roots of the G. elegans, and its expressions showed a downward trend under 4℃low temperature stress. After prokaryotic expression induction, the recombinant protein of the GeLOX1 showed a target band at approximately 111 kD, and the accumulation of the recombinant protein reached its peak at 8 h of induction. In addition, the plate stress experiment showed that the prokaryotic expression strain of the GeLOX1 was more sensitive to low temperature stress compared to the control group. The research results indicate that GeLOX1 of G. elegans can respond to low temperature stress.更多还原