Preliminary Study On Tissue Culture System Of Paris Polyphylla And Identification Of Key OSC Enzymes For Steroidal Saponin Biosynthesis

Paris polyphylla has high medicinal value,the main medicinal parts are rhizomes of Paris polyphylla Smith var.yunnanensis(Franch.)Hand.-Mazz and Paris polyphylla Smith var.chinensis(Franch.)Hara.The main medicinal component is steroidal saponin,also known as polyphyllin.But its content is very low and unstable,which makes it difficult to standardize the quality of medicinal materials.Because of the low seed germination rate,slow growth and over excavation of Paris plants in the natural environment,the resources of Paris are greatly damaged.To improve the germination rate of P.polyphylla seeds,to study the accumulation of steroidal saponins in various tissues,and to explore the key enzymes in the biosynthesis pathway of steroidal saponins in P.polyphylla will lay a foundation for the breeding of P.polyphylla seedlings,the efficient utilization of effective components,and the efficient heterologous biosynthesis of polyphyllin.In this study,the method of breaking seed dormancy of P.polyphylla var.yunnanensis was studied by temperature stratification and solution soaking.The content of polyphyllin in different tissue parts of P.polyphylla var.yunnanensis from different habitats was detected,and the accumulation rule of polyphyllin in each tissue part was determined;The transcriptome of different tissues of P.polyphylla var.yunnanensis was sequenced,and the candidate genes of biosynthesis pathway were predicted according to the accumulation of saponins in P.polyphylla var.yunnanensis;The function of OSC gene was cloned and verified.The main results are as follows:1.The seed germination of P.polyphylla var.yunnanensis was induced by stratification and solution soaking.It was found that the variable temperature deposition had no obvious effect on improving the germination rate of P.polyphylla var.yunnanensis seeds.The treatment of 2%H2O2+200 mg/L GA3 soaking solution combined with constant temperature stratification for 2 months could make the average germination rate of seeds reach 99.33%within 4 months,and the seeds released from dormancy could grow in 8 months the cotyledons were expanded within 12 months.2.After disinfection of seeds,roots,stems and leaves with 75%alcohol for 30 seconds,the contamination rate of seeds could be reduced to the lowest(0.67%)when using 15%NaClO stock solution for 40 minutes;the bacteria free survival rate could reach 60%when using 2%NaClO to disinfect roots for 9 minutes;the aseptic survival rate could reach 39.67%when using 2%NaClO to disinfect stems for 5 minutes;and the bacteria free survival rate could reach 60%when using 2%NaClO to disinfect roots for 5 minutes When the leaves were disinfected with NaClO for 7 minutes,the aseptic survival rate reached 55.67%.The obtained sterile seeds were planted on 1/2 MS medium for germination,and the germination rate was 9.2%after 4 months,and the cotyledons were expanded after 12 months;while the germinated seeds transferred to B5 medium germinated directly in the rhizome and grew into complete stems and leaves after 2 months.3.Root,stem and leaf as explants could not induce the formation of callus.After 2 months of induction and culture,the cut mature seeds could induce the formation of spherical cell mass similar to callus.4.The contents of saponins in rhizome,fibrous root,bud,stem,leaf,unripe seed,ripe seed and fruit of P.polyphylla var.yunnanensis were determined,7 kinds of polyphyllin were distributed in all tissues.The content of polyphyllin Ⅶ was relatively high in all tissues.Most of the polyphyllin were concentrated in the underground part of rhizome,fibrous root.But the content of leaf,fruit and bud is also relatively rich.The content of polyphyllin in mature fruits of P.polyphylla var.yunnanensis from Dali was rich,and the content of total saponins was the highest,among which polyphyllin Ⅶ and polyphyllin Ⅱ The content of diosgenin was significantly higher than that of the other two kinds of P.polyphylla.5.Complete the transcriptome sequencing of 8 tissues including rhizome,fibril root,stem,leaf,ripe fruit,ovary,petal and anther.The second generation transcriptome sequencing obtained 292.69 Gb of clean data,the third generation transcriptome sequencing obtained 81.81 Gb of clean data,annotated 38353 unigenes in total.The gene cluster of 7 modules was found to be related to polyphllin biosynthesis.6.According to the transcriptome data with Avena sativa L.OSC Blast analysis,will receive the candidate sequences and the known functions of OSC,evolutionary tree clustering analysis and quantity analysis,the expression of tissue eventually found 11 candidate OSC genes,tobacco use transient expression method of verification,It was found that 10 OSC genes could improve the yield of cycloatenol in tobacco,so they were identified as OSC genes.The binding ability of different OSC sequences to the substrate 2,3-squalene oxide was studied by molecular docking method.In this study,we carried out preliminary studies on the release of seed dormancy,the acquisition of tissue culture seedlings,callus induction,saponin accumulation,transcriptome analysis and key enzyme gene function of P.polyphylla,which laid a foundation for artificial breeding,efficient utilization of effective components and biosynthesis of P.polyphylla.