Identification And Functional Validation Of LncRNAs Related To Ginsenoside Synthesis

Ginseng(Panax ginseng C.A.Meyer)is a kind of perennial herb that belongs to the Ginseng genus of the Araliaceae family.It is also a kind of ancient and precious Chinese herbal medicine.Ginsenoside is one of the main active components of ginseng,which has been proved to have many medicinal values.Identification and verification of genes with important functions in the synthesis and regulation of ginsenosides are very important to clarify the synthesis and regulation mechanism of ginsenosides and improve the content of ginsenosides in ginseng and its culture.Long non-coding RNA(Lnc RNA)is a class of RNA with a length of no less than 200 nucleotides,which cannot encode protein in general.Studies have shown that Lnc RNA plays an important role in the regulation of gene expression in eukaryotes,but there are still few studies on ginseng Lnc RNA,and have no functional validation study on important Lnc RNA in regulating ginsenoside synthesis.In this study,the Lnc RNA of Jilin ginseng was comprehensively screened and analyzed,and the important Lnc RNA regulating ginsenoside synthesis was identified.Its function of regulating ginsenoside synthesis was verified by elicit regulation and Agrobacterium-mediated genetic transformation.The results obtained are as follows:(1)24,704 Lnc RNA were screened by the property analysis method and database comparison method from the Jilin ginseng transcriptome database,which forms the Jilin ginseng Lnc RNA database.(2)Through the correlation analysis between the expression of Lnc RNA in Jilin ginseng 42 farm cultivars and the content of ginsenosides,1,095 Lnc RNA were screened out due to the extremely significant correlation with the content of different mono-ginsenosides and total saponins(P-value<1.0E-02).(3)Through the association analysis between the SNP/Indels mutation of Lnc RNA obtained above with the changes in ginsenoside content,57 SNP/Indels mutations belonging to 25 Lnc RNA are significantly correlated with the changes in ginsenoside content.It is speculated that this 25 Lnc RNA may have important biological functions in the synthesis of ginsenoside.(4)Through the co-expression analysis and the co-expression network analysis conducted between 25 Lnc RNA obtained above with 17 known ginsenoside synthesis key catalytic enzyme genes,several important Lnc RNA were screened out due to the closely associated with the known ginsenoside synthesis key catalytic enzyme genes.Among these Lnc RNA,the association between Pglnc14871 and the key catalytic enzyme gene of ginsenoside synthesis is the most closely,which indicated that Pglnc14871 has an important regulatory function in ginsenoside synthesis.Therefore,Pglnc14871 is taken as the target for in-depth functional verification.(5)In the transcriptome of ginseng adventitious roots elicited by Me JA,the expression of Pglnc14871 decreased significantly compared with the control group,and its expression had an extremely significant negative correlation with the contents of various ginsenosides and total saponins.It was proved that Pglnc14871 had an important regulatory function in the synthesis of ginsenoside induced by Me JA.(6)The Pglnc14871 and its antisense RNA Anti Pglnc14871 were successfully cloned,and the engineering strain of Agrobacterium tumefaciens c58c1 containing Pglnc14871-p CAMBIA3301 and Anti Pglnc14871-p CAMBIA3301 recombinant expression vector was constructed.11 ginseng hairy-root overexpressing Pglnc14871 and 1 ginseng hairy-root antisense expressing Pglnc14871 were obtained by Agrobacterium-mediated genetic transformation.(7)Detected the gene expression and ginsenoside content of ginseng hairy-root antisense expressing Pglnc14871,the relative expression levels of Anti Pglnc14871 and ginsenoside key catalytic enzyme genes DS-1 and DS-3 were significantly higher than those in the control group,and the contents of mono-ginsenoside Rf and Rh1 were significantly higher than those in the control group.These results indicate that Anti Pglnc14871 could significantly promote the synthesis of mono-ginsenoside Rf and Rh1.It was further verified that Pglnc14871 had a significant regulatory function in ginsenoside synthesis.