目的 克隆茅苍术Atractylodes lancea细胞分裂素羟化酶基因（AlCYP735A，GenBank：PP839072.1），对其进行生物信息学分析、组织特异性表达分析以及构建原核表达载体对该蛋白进行重组表达。方法 从茅苍术cDNA中克隆AlCYP735A序列全长，利用生物信息学方法分析其编码蛋白的理化性质，qRT-PCR检测其组织特异性表达情况，连接pET28a-mbp表达载体转入大肠杆菌BL21进行重组蛋白的表达，并在烟草叶片中对其进行了亚细胞定位。结果 AlCYP735A基因的开放阅读框（open reading frame，ORF）长度为1 566 bp，编码521个氨基酸；拥有1个P450基因保守域，相对分子质量为59 328.6，等电点为9.13，有信号肽，属于分泌蛋白。qRT-PCR结果表明其根茎中表达量最高，与其他组织器官具有较明显差异。重组蛋白mbp-AlCYP735A获得可溶性表达，大小符合106 700的预期。烟草亚细胞定位显示其定位在叶绿体。结论 成功克隆AlCYP735A的基因，完成生物信息学分析、探明组织特异性表达情况和在烟草中的亚细胞定位，重组蛋白在原核表达系统中获得了可溶性表达，为后续该基因的功能验证提供了条件。

Objective The gene AlCYP735A (GenBank: PP839072.1), coding for cytokinin hydroxylase in Atractylodes lancea was cloned for bioinformatic analysis, tissue-specific expression analysis, and recombinant expression using a prokaryotic expression vector. Method The full length of AlCYP735A was cloned from the cDNA of A. lancea, and its physicochemical properties were analyzed using bioinformatics tools. The tissue-specific expression of this gene was examined using qRT-PCR, and it was ligated into the pET28a-mbp expression vector and transformed into E. coli BL21 for expression of the recombinant protein. The subcellular localization prediction was carried out in tobacco leaves. Result The open reading frame of AlCYP735A is 1 566 bp, encoding 521 amino acids. It has a conserved P450 domain, with a relative molecular weight of 59 328.6 and an isoelectric point of 9.13. It contains a signal peptide and is classified as a secretory protein. The qRT-PCR results indicated that its expression is highest in the rhizome, significantly different from other tissues and organs. The recombinant protein mbp-AlCYP735A was solubly expressed, with a size consistent with the expected 106 700. Tobacco subcellular localization shows its localization in chloroplasts. Conclusion The successful cloning of the AlCYP735A gene, completion of bioinformatic analysis, determination of tissue-specific expression, soand subcellular localization in tobaccoluble and expression of the recombinant protein in the prokaryotic system provide the groundwork for subsequent functional validation of this gene.

R286.12

湖北省教育厅青年项目（Q20222013）；湖北省科学技术厅青年项目（2022CFB576）