| Pueraria thomsonii Benth is an important herb medicine and starch plant,its medical function includes anti tumor,lowering blood pressure and blood lipid.It can be used to make types of food such as Pueraria Powder,Pueraria Tablets.In recent years,the growing areas of P.thomsoniiis had being spreaded and cultivation acreage increased rapidly.This study did research in three aspects with a major cultivar in Hubei province En Ge 08.1)Identification of secondary metabolites in tuber root of P.thomsonii;2)Genes involved in secondary metabolites biosynthesis and starch were studied in transcriptome analysis,which paved foundation for improving medical material accumulation.3)The cultivation conditions combined NPK fertilizer,planting density,lighting and soil water content were optimized to increase the yield of P.thomsonii and the accumulation of medicinal substances,and the best harvest time was determined according to the accumulation dynamics.The type of medicinal substances contained in P.thomsonii metabonomics of P.thomsonii tuber was analyzed.Twelve compounds were isolated from the extract of P.thomsonii tuber by silica gel column chromatography and semi preparative high performance liquid chromatography.Their structures were elucidated by HR-MS,1D and 2D nuclear magnetic resonance(NMR)data.Seven compounds were isolated from P.thomsonii for the first time,including p-coumaric acid,methyl coumaric acid,(E)ethyl p-coumaric acid,4"-hydroxyisoflavone,glycyrrhizin,benzoic acid,5,7-dihydroxy-3-[4'-O-(3-methyl-2-butenyl)-phenyl]-isoflavone.Six compounds,including 4"-hydroxyisoflavone,benzoic acid,5,7-dihydroxy-3-[4'-O-(3-methyl-2-butenyl)-phenyl]-isoflavone,were isolated from Pueraria for the first time,which were significantly different from the chemical constituents identified in the literature.The contents of puerarin,daidzein,daidzein and chickpea sprout a in different tissues of P.thomsonii were different.The study on the regulation of metabolite gene expression in P.thomsonii is helpful to explore the genes related to metabolite biosynthesis.Transcriptome analysis of leaves,stems and roots of P.thomsonii showed that 44,339 non redundant transcripts were obtained,of which 43,195 were functional annotated.The metabolic pathway of isoflavones in P.thomsonii was inspected,and the structural genes involved in the synthesis of isoflavones and starch were identified.Compared with the gene expression level of tuber root,9225 deferentially expressed genes were identified,including 32structural genes potentially involved in Isoflavone Biosynthesis.q RT-PCR was used to detect the expression of 8 genes involved in flavonoid,isoflavone and starch synthesis.The results showed that the expression of dihydroflavonol 4-reductase and other genes increased in P.thomsonii.Further analysis showed that 437 transcription factors may be involved in the regulation of isoflavone biosynthesis.micro RNAs are involved in defense response.Mir156a in Pueraria may promote Isoflavone Biosynthesis by inhibiting SBP;Mir319may promote isoflavone biosynthesis by inhibiting TCP and hb-hd-zip.The calli culture system of P.thomsonii was established.CRISPR/cas9 vector was constructed and introduced into calli of P.thomsonii.The function of F01.pb15220 gene was verified by gene editing.Under the optimized culture conditions,the expression level of the gene was increased and the accumulation of Puerarin was increased,which indicated that the gene was involved in puerarin biosynthesis pathway and regulated by NAA.Objective to explore the best cultivation conditions for increasing the yield and medicinal components content of P.thomsonii.The results show that P.thomsonii is a kind of crop which likes potassium,partial phosphorus,light nitrogen,light and waterlogging.The best concentration of urea,calcium superphosphate and potassium sulfate were 0.5%,0.75%and 0.75%,respectively;The best planting density was 950plants/667m~2(plant row spacing 70 cm)×100 cm);The conditions of protected cultivation of P.thomsonii were discussed,and the effect of lighting of 3000 lux(LX)in greenhouse was the best;the most suitable soil moisture content in greenhouse is35-40%.According to this study,the total biomass,weight of P.thomsonii,starch accumulation,total isoflavones and other medicinal substances accumulate similarly as food material.P.thomsonii can produce the highest yield in December,but as a medicinal material,it can get the highest content of medicinal substances in October.In summary,the species and accumulation of medicinal compounds in the metabolites of P.thomsonii were analyzed by means of histochemistry;The transcriptional analysis identified the isoflavones and the molecular mechanism of their synthesis,which laid a theoretical foundation for molecular improvement of Pueraria;The optimization of cultivation conditions provides a reliable basis for the high yield and high quality production of P.thomsonii. |
PDF Full Text Request