Establishment Of Genetic Transformation System Of Hairy Roots And Study On Biosynthesis Mechanism Of Plumbagin In Plumbago Auriculata Lam.

As a multi-functional garden ornamental and medical plant,Plumbago auriculata has been paid more and more attention in garden-plant landscaping and medical commercial developments and appllications due to its excellent ornamental value and a large number of secondary metabolites plumbagin accumulated mainly in roots.In recent years,it was an important means for producing of plant secondary metabolites by in vitro culture of hairy roots in biological reaction systems.Studies on the regulation of plant secondary metabolites and their biosynthesis mechanisms are still hot topics in academia.To date,some questions have no satisfactory answeres,and there still lack targeted regulatory techniques and biosynthetic pathways in molecular biology for the specific plants,such as P.auriculata in this study.However,there is no literature on the regulation mechanism of hairy roots and plumbagin biosynthesis in P.auriculata,and the genes related to the regulation of plumbagin synthesis and its metabolic pathways are not clear at present.Therefore,firstly the genetic transformation system of hairy roots was established in this study,and based on it elicitors was used to regulate the plumbagin biosynthesis.Then,the RNA-seq technique was used to compare the gene expression levels under different physiological conditions,and key genes involved in the plumbagin biosynthesis pathway was selected for functional verification and providing the possibility to study the construction of secondary metabolic regulation network of plumbagin.The results of this study filled the molecular biology research gap of plumbagin synthesis pathway.It also laid an important theoretical foundation for the protection of the wild germplasm resources and the development of gene resources related to plumbagin synthesis and subsequent molecular breeding in P.auriculata.In this study,the main findings obtained are as follows:1)A hairy root induction and culture system of P.auriculata was successfully established for the first time.Seeds of P.auriculata germinated well on hormone-free MS solid medium and a large number of high quality sterile seedlings were obtained.Three strains of Agrobacterium rhizogenes,A4,ATCC 15834 and LBA 9402,all could induce hairy roots of sterile leaf explants.Of which ATCC 15834 had the strongest rooting ability and the earliest rooting time（8.33±0.58 d）,the highest induction rate（86.78±0.74%）,while the browning rate（2.07±0.32%）,callus rate（2.04±0.08%）,pollution rate（0.11±0.02%）and mortality rate（5.43±0.69%）were the lowest than the LBA 9402 and A4.All hairy root lines could rapidly grow and subculture on hormone-free 1/2MS medium.The colour was white or milk-white,with typical hairy root morphology such as minuteness,root hairs,multi-branched,and apogeotropism.Paraffin sections showed that the significant difference in the cell structure between hairy roots and tissue culture roots was root hairs,indicating that the hairy roots were real.PCR confirmed that the Ri plasmid on A.rhizogenes had integrated into the leaf explant genome and presented the corresponding biological phenotype of hairy roots,and further explained the authenticity of the hairy roots.The whole process did not require pre-culture,and directly co-culturing for 2 to 3 days was most beneficial to the induced transformation of the hairy roots in P.auriculata.2)Hairy roots produced by genetic transformation system in P.auriculata were effective,and the plumbagin content was significantly higher than that of the control group（tissue culture roots and plants roots）.Under the same culture conditions,plumbagin content in the hairy root lines（30 d）was PAHR 15834>PAHR 9402>PAHR 4,and it in PAHR15834 was 38.95 mg·g^–1DW or 3.90%DW.In the study on growth kinetics for 60 d,the growth patterns of the three hairy root lines were all typical“S”curves,and PAHR 15834was the most effective in both biomass growth index and plumbagin accumulation stability.PAHR 15834 biomass growth index was divided into four stages,the lag phase（0-6 d）,the exponential phase（6-30 d）,the stable phase（30-36 d）and the decline phase(after the 36^thd).The synthesis and accumulation of plumbagin also included for stage,the synthetic initial period（0-6 d）,the synthetic exponential phase（6-33 d）,the synthetic stable phase（33-36 d）,and the late synthetic phase(after the 36^th d).The improved HPLC method explored in this study had high precision,linearity,stability and repeatability for the determination of plumbagin content.3)Both elicitors Me JA and SA could further promote the accumulation of plumbagin in the hairy roots.The Me JA(10,50,100μmol·L^–1)and SA(10,50,100 100μmol·L^–1)all increased the plumbagin content in hairy roots.Me JA 100μmol·L^–1had the highest content of 8.42±0.01%DW at the 30^th d,which was 1.78 times that of the control group at the same day.While SA 50μmol·L^–1was followed（5.98±0.02%DW）at the 30^th d,which was1.27 times that of the control group at the same day.In addition,plumbagin content of each time（0 h,6 h,12 h,18 h,24 h）at the 0 to the 24^th h was significant changed during the whole treatment progress of 30 d,and it reached 3.89±0.03%DW,4.65±0.03%DW,5.74±0.02%DW,6.99±0.02%DW,7.48±0.04%DW,respectively.4)Based on the plumbagin content significantly increased after treatment by Me JA100μmol·L^–1,samples obtained at four time-point were used for RNA-seq.A total of 46,221 Unigenes were obtained,of which 25,400 Unigenes were annotated.A total of 10,528up-regulated genes and 16,355 down-regulated genes were obtained by differentially expressed genes analysis.The functional annotation found that P.auriculata had the highest homology with three species,namely Beta vulgaris,Vitis vinifera and Theobroma cacao.For GO classification,the most involved processes were metabolic processes,cellular processes and single biological processes in the biological process,cells,cell parts and organelles in the cell composition,and catalytic activity,binding and transport activity in the molecular function.For KOG database predicts,26,467 genes would be classified into25 functional categories.For KEGG database alignment,5,877 Unigenes were annotated into 113 pathways,among which Unigenes in metabolic pathways were the most（42.1%）,followed by the secondary metabolite biosynthesis pathway（21.51%）.5)Based on the results of RNA-seq,7 candidate differential genes（MENG,PKSB,PRS4,pgk2,PK and 2 PFK3）involved in plumbagin biosynthetic pathway of hairy roots and 1 internal reference gene（ACTIN）were selected for q PCR analysis.Except for 1 PFK3（Unigene0039043）,the trend of q PCR expression of other candidate genes was consistent with the trend of RNA-seq expression,indicating that these genes may play a key regulatory role in the plumbagin biosynthesis of hairy roots in P.auriculata.Preliminary speculation of the route of plumbagin synthesis was mainly glycolysis and tricarboxylic acid cycle.Especially,acetyl-Co A was catalyzed by PSKB to form polyketide,and the production was further catalyzed by MENG to form naphthoquinone（plumbain,Figure 6-4）.