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目的:克隆香叶醇-10羟化酶(geraniol 10-hydroxylase,G10H)基因,进行生物信息学分析,并在不同广藿香栽培种中分析不同时期的茎、叶的表达情况。方法:搜索广藿香转录组数据库,获得G10H基因全长序列,并设计全长引物进行PCR验证;利用生物信息学软件对PcG10H1基因进行生物信息学分析,实时定量PCR法对其进行了时空表达分析。结果:将获得的广藿香香叶醇-10羟化酶基因命名为PcG10H1(Gen Bank登录号为KF926077),该基因包含一个完整的长1 533 bp的开放阅读编码框,编码510个氨基酸。分析了PcG10H1基因编码蛋白的理化特性。分析发现其有1个跨膜区,无信号肽;PcG10H基因编码的氨基酸序列与胡黄连G10H基因编码的氨基酸序列最为接近;PcG10H1在老叶和老茎中表达量高,在老叶中表达量最高;从不同栽培种来看,PcG10H1在石牌广藿香和高要广藿香中表达模式相似,在海南广藿香与印尼广藿香中表达相似。结论:成功克隆了广藿香PcG10H1的全长序列且进行了序列分析,并对其在不同栽培种的表达模式进行了探讨,为进一步阐明广藿香萜类代谢途径奠定基础。
OBJECTIVE: To clone the geraniol 10-hydroxylase (G10H) gene for bioinformatics analysis and analyze the expression of stem and leaf at different stages in different patchouli cultivars. Methods: The genomic DNA of Patchouli was searched for the full-length G10H gene sequence and the full-length primers were designed for PCR validation. The bioinformatics software was used to analyze the PcG10H1 gene in a spatial-temporal analysis by real-time quantitative PCR analysis. Results: The gene of patchoulen geraniol-10 hydroxylase was named as PcG10H1 (Gen Bank accession number KF926077). The gene contains a complete 1 533 bp open reading frame coding for 510 amino acids. The physicochemical properties of PcG10H1 gene were analyzed. The results showed that PcG10H gene had the closest transmembrane domain and no signal peptide. The amino acid sequence of PcG10H gene was closest to the amino acid sequence encoded by G10H gene of P. hupehensis. The expression of PcG10H1 in old leaves and old stems was high, Highest; from different cultivars, PcG10H1 expression pattern in Patchouli and Patchouli are similar, and Patchouli in Hainan and Patchouli in Indonesia are similar. Conclusion: The full-length sequence of PcG10H1 was successfully cloned and sequenced. The expression patterns of PcG10H1 in different cultivars were explored, which laid the foundation for elucidating the metabolic pathway of terpineoids in patchouli.