目的 从广藿香Pogostemon cablin中克隆HAD（haloacid dehalogenase）超级家族蛋白水解酶基因PcHAD，验证其编码蛋白与广藿香醇合酶（patchoulol synthase，PcPTS）蛋白互作，探究其参与调控广藿香醇生物合成的作用。方法 从广藿香转录组数据中，筛选出一条具有完整编码区并且包含HAD-like结构域的HAD基因序列（PcHAD），并对该基因进行克隆和生物信息学分析。利用qRT-PCR对PcHAD的表达模式进行分析。利用酵母双杂交实验进一步验证PcHAD与广藿香PcPTS蛋白的互作情况。通过瞬时过表达PcHAD，分析其对广藿香醇甲羟戊酸合成途径（mevalonate pathway，MVA）相关基因的表达以及对广藿香醇含量的影响。结果 从广藿香中克隆得到HAD-like基因PcHAD，其开放阅读框为1149 bp，编码蛋白长度为382个氨基酸，蛋白相对分子质量为43 000，且为不稳定的亲水性蛋白，亚细胞定位结果表明该蛋白定位于叶绿体。酵母双杂交实验结果表明PcHAD完整蛋白能与PcPTS完整蛋白互作，且PcHADN与PcPTSN互作。与对照组相比，瞬时过表达PcHAD广藿香叶中的广藿香醇含量提高了30%，MVA途径基因包含PTS、FPPS和HMGR在内表达量均显著上调。结论 从广藿香中克隆得到1个与广藿香醇合酶PcPTS互作的PcHAD基因，该基因在瞬时过表达后上调PTS和MVA途径多个基因的表达量，且能够显著提高广藿香醇的含量，表明该基因可能在广藿香醇生物合成中发挥正向调控作用，为进一步揭示广藿香中HAD-PTS复合体的功能提供科学依据。

Objective PcHAD gene of HAD (haloacid dehalogenase) superfamily protease was cloned from Guanghuoxiang (Pogostemon cablin), and its coding protein interaction with patchoulol synthase (PTS) protein was verified to explore its role in regulating the biosynthesis of patchouli alcohol. Methods In this study, a HAD gene sequence (PcHAD) with complete coding region and HAD-like domain was screened from P. cablin transcriptome data for cloning and bioinformatics analysis. The tissue expression pattern of PcHAD was analyzed by RT-qPCR. The yeast two-hybrid experiment was used to verify the interaction between PcHAD and PcPTS protein. Transient overexpression of PcHAD was used to analyze the effect of PcHAD on the expression of genes related to the MVA pathway and the content of patchouli alcohol. Results The HAD-like gene PcHAD was cloned from P. cablin. Its open reading frame was 1149 bp, encoding 382 amino acids, and the molecular weight of the protein was 43 000. It was an unstable hydrophilic protein and subcellularly localized in the chloroplast. In the yeast two-hybrid assay, the PcHAD protein can not only interact with the PcPTS protein, but also with PcPTSN. After transient overexpression of PcHAD, the content of patchouli alcohol was increased by 30% compared with the control group, and the expression of genes on the MVA pathway including PTS, FPPS and HMGR were significantly up-regulated. Conclusion In this study, HAD gene that interacts with patchoulol synthase PcPTS was cloned from P. cablin. Transient overexpression of PcHAD can increased the content of patchouli alcohol and the expression levels of PTS and genes involved in the MVA pathway, suggesting that this gene may play a positive role in the biosynthesis of patchouli alcohol. These results provided scientific basis for further research on the function of HAD-PTS complex in P. cablin.

R286.12

国家自然科学基金资助项目(81803657);广东省自然科学基金资助项目(2019A1515011542);广东省重点领域研发计划(2020B020221001)