| Patchouli alcohol is one of the main active ingredients in patchouli oil,has been the index component for evaluating patchouli medicinal materials and patchouli oil quality in different versions of Chinese Pharmacopoeia.Patchouli alcohol has been widely used in pharmaceuticals and cosmetics,because of its obvious antibacterial and antiviral activities and unique pleasant smell.Pogostemon cablin(Blanco)Benth.is a major biological resource to extract the patchouli alcohol,but its further development and utilization are limited because of the lower content of patchouli alcohol in the patchouli oil.Therefore,it is necessary to study the biosynthesis of patchouli alcohol.The content of patchouli alcohol in leaves is higher than other parts,and there is a visible difference between the initial growth stage and maturation stage.The transcriptomic analysis was performed to compare the differentially expressed genes(DEGs)in the seedling stage(about 60 days after cutting)and maturation stage(about 240 days after cutting),our research has found three key enzyme gene of patchouli alcohol synthesis,Famesyl diphosphate synthase(FPS),Patchoulol synthase,(PTS),and HMG coenzyme A reductase,(HMGR)showing a different expression level.We have obtained the conserved sequence of FPS by the PCR amplification technique,and carry out bioinformatics analysis.Real-time PCR was applied to analyze the relative quantitative expression of two growth and development stages.The main results were as follow:(1)The essential oil of P.cablin leaves were extracted by steam distillation method,and analyzed by GC-MS technique.The contents of essential oi in seedling stage and maturation stage were 0.55%and 1.13%,respectively,showed significant difference.The contents of patchouli alcohol in essential oil at maturation stage was increased 10.43%compared with the seedling stage.Meanwhile the contents of other sesquiterpene compound were improved in different extent.(2)RNA-Seq was based on the leaves of P.cablin in seedling stage and maturation stage,which was used to obtain an average length of 90 nt and the total of 63.7 million and 65.9 million clean reads,respectively.162,509 unigene were annotated by using BLAST against NR(NCBI non-redundant protein sequences),KOG(Eukaryotic orthologues groups),GO(Gene ontology),KEGG(Kyoto encyclopedia of genes and genomes)databases after De novo assembling.105,430 unigene blast against NR,the most similarly was 69.87%with Sesamum indicum.Compared with the KOG database,the 83,369 unigene were divided into 25 classes according to the function.12,261 unigene annotated against GO database were divided into 3 categories with a total of 49 functional groups.KEGG pathway analysis showed that 79,053 unigene could be divided into 124 classes,and partial unigenes were involved in biosynthesis of secondary metabolites,sesquiterpenoid and triterpenoid biosynthesis,flavone and flavonol biosynthesis,anthocyanin biosynthesis and so on.(3)The sequence of FPS obtained by the PCR amplification technique was 1177 bp,this sequence contain a length of 1050 bp open reading frame(ORF),the enzyme consists of 349 amino acid,the similarity is 93.10%compared with other plants(Lamiaceae),FPS belong to Isoprenoid Biosynthesis C1 superfamily.FPS protein was predicted that the molecular mass was about 40.09 kD,the pI was about 5.34.This enzyme was hydrophilic,which also contained no transmembrane and signal peptide.(4)Differential expression levels of FPS.PTS and HMGR gene were analyzed by Real-time PCR.The results showed that the relative quantitative expression in maturation stage was higher than seedling stage of P.cablin leaves,this was consistent with the result of transcriptomic analysis.The information generated from this study provides a solid base for further study of the molecular mechanism of patchouli alcohol biosynthesis,and the differentially expressed genes(DEGs)in this biosynthetic pathway,and finding the key enzyme implicated in the regulation of the pathway. |
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