Primary Study On The Cloning And Functioning Of 8-HGO Transgene In Lithospermum Erythrorhizon

Shikonin and its derivatives(hereafter shikonin)are red extracts from the roots of Lithospermum.The main components are naphthoquinones,which can be used not only for treating dermal disease like wounds,burns,hemorrhoids,etc.,but also have anti-inflammatory,anti-viral,anti-tumor and other function.shikonin and its derivatives have great market value and application prospects,so the research on their synthesis and regulation is such a great breach.At present,two main upstream biosynthetic pathways of shikonin,Mevalonate(MVA)pathway and Phenylpropanoids(PP)pathway have been fundamental to master,but some of reactions downstream are still unclear,especially the mechanism involved in the transformation from 3'-hydroxy-geranylhydroquinone to shikonin.In the previous transcription study,a gene called 8-HGO was found,its gene expression level was weak in growth medium B5 but higher in the synthetic medium M9 of shikonin.a great deal of researches showed that 8-HGO enzyme genes encodes 8-hydroxygeraniol dehydrogenase(8-HGO),which plays an essencial role as a dehydrogenase in many iridoid synthesis of secondary metabolites.Therefore,this experiment attempts to clone the gene and explore the function of 8-HGO in the regulation of the biosynthesis of shikonin and its derivatives.Based on the transcriptome sequencing results of Lithospermum,we obtained the original 8-HGO c DNA sequence,and analyzed the physicochemical properties of 8-HGO protein using bioinformatics software to predict its secondary and tertiary structure.The results showed that the full-length ORF of 8-HGO was 1083 bp,which could encode 360 amino acids and was a medium chain Zn-dependent alcohol dehydrogenase.We designed primers,cloned the target gene,and constructed the overexpression and RNAi vectors using the p BI121-e GFP plasmid as the vector,and finally introduced them into Agrobacterium rhizogenes ATCC15834 to infect explants.Finally,we introduced several overexpression of transgenic hairy roots(8-HGO-O,HO),RNAi transgenic hairy roots(8-HGO-RNAi,Hi),and compared hairy root(Empty vector,EV),each with no less than 6 strains.after collecting shikonin of M9 culture solution and hairy roots to determine the OD520 value,we calculated the content of shikonin,and use HPLC to detect various hairy root product components.Finally,detecting the target gene and related regulatory genes to further analyse the function of 8-HGO.In order to verify the hairy roots,first,the rol C and GFP genes in each hairy root were detected by PCR,and then observed with a laser confocal microscope,It found that the transgenic hairy roots of EV,HO,and Hi were fluorescent,which further proved that they were 8-HGO transgenic hairy roots.And It turned out to be that we provides particularly critical and important experimental materials for exploring 8-HGO in the synthesis and regulation of secondary metabolites of Lithospermum.The hairy roots of HO,Hi and EV were cultured in M9 medium for 6 days.The M9 culture solution of the hairy roots of HO was the darkest purple,and the colors of Hi and EV were similar.The HPLC chart showed that the main products secreted by the hairy roots in the M9 culture solution were shikonin and acetylshikonin.The 8-HGO gene expression of the hairy roots of each strain was detected by q PCR.The results showed that compared with EV,8-HGO expression was highly expressed in HO and suppressed in Hi.Which means the overexpression of HO hairy roots is obvious and the RNA interference of Hi-8 is feasible.after calculating and comparing present the content of shikonin,the data displayed the overexpression of 8-HGO could improve the yield of shikonin.What's more,the expression levels of shikonin-related synthesis genes in 8-HGO transgenic hairy roots preliminarily showed that 8-HGO may have little impact on PGT,PAL and HMGR.To sum up,the tentative detection of the role in the regulation of shikonin synthesis of 8-HGO from many aspects provides the necessary first experiments and theories for the regulation of Lithospermum's secondary metabolism,but the specific mechanism needs more experiments to explore.