Cloning、Bioinformatics And Expression Analysis Of HsFPS1、HsSQS1and HsSE1in Huperzia Serrata

Shezushishan is the dried whole plants of [Huperzia serrata (Thunb.) Trev.] that belongs to the family of Huperzia. It is one of the most commonly encountered traditional Chinese herbal medicines for the treatment of contusions, strain, swelling and schizapophrenia. The main chemical constituents of this plant were Lycopodium alkaloids, terpenoid and flavonoid. Huperzine A, which belongs to the lycodine type of lycopodium alkaloids, has been used as an anti-Alzheimer’s disease drug candidate. Besides lycopodium alkaloids, terpenoids have been tested to possess many bioactive activities. However, the biosynthetic pathway of terpenoid remains unclear in H.serrata. Based on the knowledge of terpenoid biosynthesis, the genes encoding key enzymes of farnesyl pyrophosphate synthase (FPS)、squalene synthase (SQS) and Squalene epoxidase (SE) are involved in the biosynthetic pathways of plant triterpenoid and phytosterols. Our research group obtained a great number of transcript from the transcriptome dataset of H.serrata and found many candidate genes might be involved in terpenoid biosynthesis.Based on the above research results, this study completed the following research contents:After searching the transcriptome dataset of H.serrata, three unique sequence encoding FPS1、SQS1and SE1were discovered. The primers were designed according to the transcript sequence of FPS1、SQS1and SE1from the H.serrata transcriptome dataset. And then, the open reading frame of three genes was cloned using RT-PCR strategy from H.serrata. The bioinformatic analysis of three genes and the corresponding protein was performed.The cDNA (named as HsFPS1) contains a1,119bp open reading frame and encodes a predicted protein of372amino acids, with a calculated molecular Weight of 42.68kDa and an estimated PI of6.60. The GenBank accession number for this gene is JX027508. No transmembrane regions and signal peptide was presented in HsFPSl. The conserved domain of Terpenoid synthase was presented in HsFPSl. HsFPSl was more abundant in H.serrata root than in leaf and stem.The cDNA (named as HsSQSl) contains a l,263bp open reading frame and encodes a predicted protein of420amino acids, with a calculated molecular Weight of47.76kDa and an estimated PI of6.60. The GenBank accession number for this gene is JQ004938. HsSQS1contains two transmembrane regions, without signal peptide. The conserved domain of squalene synthase was presented in HsSQSl. HsSQSl was more abundant in H. serrata root than in leaf and stem.The cDNA (named as HsSEl) contains a l,686bp open reading frame and encodes a predicted protein of561amino acids, with a calculated molecular Weight of62.78kDa and an estimated PI of9.91. The GenBank accession number for this gene is JX027509. HsSEl contains three transmembrane regions, without signal peptide. The conserved domain of Squalene epoxidase was presented in HsSEl. HsSEl was more abundant in H.serrata stem than in root and leaf.This study cloned and analyzed farnesyl pyrophosphate synthase (FPS), squalene synthase (SQS) and squalene epoxidase (SE) gene from H.serrata for the first time. The result will provide a foundation for exploring the mechanism of terpenoid biosynthesis in H.serrata plants.