Study On Cloning Squalene Epoxidase Gene Of Polygala Tenuifolia Willd. And Its RNA-seq

Objectives Cloning Squalene Epoxidase gene and conducting RNA-Seq study in Polygala tenuifolia,to provide reference for the improvement of the germplasm resources of P.tenuifolia and the biosynthesis of saponins in P.tenuifolia.Methods(1)RT-PCR and RACE technology were used to clone full-length Squalene Epoxidase gene of P.tenuifolia.And the bioinformatics analysis of the full-length Squalene Epoxidase gene and its encoded amino acid sequences were carried out.(2)RT-q PCR and HPLC were adopted respectively to determine relative expression of Squalene Epoxidase gene and tenuifin content in different parts of P.tenuifolia(root,stem and leaf)and were analyzed statistically to explore the relationship between them.(3)RNA-Seq sequencing was used to annotate structure and function of transcript and gene level data of P.tenuifolia Willd,study the triterpenoid saponin biosynthesis pathway and its backbone modification of P.tenuifolia,and screen the key enzyme genes involved in the metabolic process.Results(1)The c DNA full-length sequence(1652 bp)of the Squalene Epoxidase(SE)gene of P.tenuifolia was cloned,containing 28 bp 5'UTR,37 bp3'UTR and 1587 bp ORF region and encoding 528 amino acid protein.And the Gen Bank accession number of SE gene was MG917041.The BLAST analysis showed that P.tenuifolia SE gene was highly homologous to the other species that had been registered in Gen Bank.The relative molecular weight and isoelectric point of the protein encoded by P.tenuifolia SE gene were 57882.34 Da and 8.77 respectively.The protein belongs to PLN02985(squalene monooxygenase)and FAD-binding-3 superfamily,containing FAD-binding sites,and a highly conserved domain of SE.(2)The results of RT-q PCR showed that P.tenuifolia SE gene expressed in various parts,but the expression levels was significantly different.The expression was highest in the root,followed by the leaf.And the expression in the stem was lowest.HPLC results showed that tenuifolin content in root was significantly higher than leaf and stem.The statistical analysis revealed that tenuifolin content significantly had a positive relationship with the expression of SE gene,indicating that P.tenuifolia SE gene play a role in the accumulation of tenuifolin ingredients.(3)The transcriptome sequencing(RNA-Seq)of m RNA was performed,and in the NR database annotation the results showed that P.tenuifolia sequence could macth to many species,and the similarity was very high.The GO annotation results showed that there were 207180,144773 and 67358 unigene were classified as BP,CC and MF respectively.The results of KEGG analysis showed that 174 unigenes could be annotated to the pathway of triterpenoid saponin skeletons and 32 unigenes could be annotated to the pathway of triterpenoid saponin biosynthesis,which were the most.Further analysis of the two pathways revealed the key enzyme genes involved in the triterpenoid saponin pathway and some genes that could be annotated in this RNA-seq.Conclusions P.tenuifolia SE gene has been cloned successfully by RACE medthod.The correlation analysis between SE gene and tenuifolin content was also performed,the results revealed that tenuifolin content significantly had a positive relationship with the expression of SE gene.Forthermore,RNA-Seq was helpful to understand the complex biological functions of triterpenoid saponins,such as metabolic pathway,genetic information transmission,etc.,could screen the key genes in the triterpenoid pathway of P.tenuifolia and discover the candidategenes related to medicinal ingredients,laying down a foundation for in-depth analysis of triterpenoid pathway,key enzyme gene mining and further genetic engineering of triterpenoid biopathways in P.tenuifolia Willd..