竹节参鲨烯环氧酶基因的克隆及生物信息学分析

目的 克隆竹节参Panax japonicus鲨烯环氧酶基因（squalene epoxidase,PjSE）的全长cDNA序列，进行生物信息学分析和原核表达。方法 设计特异性引物，从竹节参中克隆得到PjSE序列；以PjSE基因序列作为输入数据，利用多序列同源比对等生物信息学分析工具进行序列分析；构建重组原核表达载体pCold-PjSE，转化至大肠杆菌BL21(DE3)中进行蛋白诱导表达；采用实时荧光定量PCR技术分析该基因在竹节参不同组织中的相对表达量。结果 PjSE基因开放阅读框长度为1884 bp，编码627个氨基酸，PjSE蛋白相对分子质量为68 639.49，初步预测其具有跨膜结构域，可能定位在叶绿体上；聚丙烯凝胶电泳结果显示诱导表达蛋白的相对分子质量大小与预期结果一致；该基因在竹节参叶中表达量最高，须根次之，根中表达量最低。结论 PjSE基因的克隆、生信分析及原核表达研究，不仅有助于丰富竹节参中该类功能蛋白的种类和数量，完善竹节参皂苷类成分的生物合成途径，也为后期开展竹节参皂苷的异源合成提供了可供选择的关键基因元件。

Cloning and bioinformatics analysis of PjSE from Panax japonicus

Objective To clone the full-length cDNA sequence (squalene epoxidase, PjSE) of Zhujieshen (Panax japonicus), and perform bioinformatics analysis and prokaryotic expression. Methods Specific primers were designed to clone the cDNA sequence of PjSE from P. japonicus. Using the PjSE gene sequence as input data, sequence analysis was performed using bioinformatics analysis tools such as multiple sequence homology alignment. The prokaryotic expression vector of pCold-PjSE was constructed and transformed in Escherichia coli BL21 (DE3) competent cells to express recombinant protein. The relative expression of PjSE in different tissues of P. japonicus were further determined by quantitative real-time PCR (qRT-PCR). Results The open reading frame of PjSE was 1884 bp, encoding 627 amino acids. The relative molecular mass of PjSE was 68639.49. PjSE was predicted to have a transmembrane domain that was most likely to be located in chloroplast. SDS-PAGE showed the relative molecular mass of induced expression protein was consistent with expected protein size. Furthermore, the expression level of PjSE was completely different in various parts of P. japonicus. The associated results were as follows: the highest in leaves, the second in fibrous root, and the lowest in root. Conclusion The cloning, bioinformatics analysis and prokaryotic expression of PjSE will be helpful to enrich the variety and quantity of related functional proteins from P. japonicus, supplement to the biosynthetic pathways of chikusetsusaponins, and provide useful functional gene modules for heterologous biosynthesis in the future.