Bioactivity And Molecular Mechanism Of Ginsenoside From The Stems And Leaves Of Panax Ginseng Against Asiatic Corn Borer,Ostrinia Furnacalis

Ginseng belongs to Araliaceae,and its main active ingredient is ginsenoside.Ginsenoside content is different among the parts of a ginseng plant.Some researchers reported that the ginsenoside content was higher in stems and leaves than roots of ginseng.The ginsenoside capsules and tablets with various biological functions were produced with ginseng stems and leaves which were not utilized traditionally.With the studies of ginseng autotoxicity,we found that ginseng stems and leaves,especially those of the wild and perennial ginseng plants,were not damaged by pest.The possible reason for the anti-insect function was that ginseng stems and leaves produced the triterpenoid saponin during ginseng growth.However,it was not clear that if there is numerical relationship between anti-insect function and ginsenoside content in ginseng stems and leaves,or which ginsenoside has the main effect on pest and what is the inner regulating mechanism.Therefore,ginseng stems and leaves were used as the experimental material,and the Asiatic corn borers were used as the experimental pest.The dissertation research simulated the natural growth environment of Asiatic corn borers through an artificial climate chamber,determined the biological activity of ginsenoside on Asiatic corn borers,studied the inhibiting effect of ginsenoside on the growth and population of corn borer larva,analyzed the effect of ginsenoside on the nutrient substances and metabolic enzymes of corn borers,elucidated the molecular mechanism of anti-insect function in ginseng with the transcriptome sequencing technology.The major results of this research are presented as follows:1.The ginsenoside in ginseng stems and leaves possessed significant killing effect on the eggs of Asiatic corn borers.LC₅₀0 of ginsenoside in stems and leaves was 48.69 mg mL-1,57.87mg mL^-1,and 69.98 mg mL^-1 in the body of 0-instar,1^st-instar,and 2^nd-instar corn borer eggs,respectively.The killing rates of 0-instar,1^st-instar,and 2^nd-instar corn borers were 73.07%,66.45%,and 60.73%when the ginsenoside concentration of 100 mg mL^-1 was used.The ovicidal activity of Panaxadiol Saponin（PDS,48.30%）was significantly higher than that of Panaxtrol Saponin（PTS,36.93%）;LC₅₀0 of PDS and PTS was 25.73 mg mL^-1 and 58.26 mg mL^-1,respectively,in the body of the 0-instar corn borer eggs.2.The ginsenoside in ginseng stems and leaves had significant antifeeding effect on the3^th-instar corn borer larva.Non-selective AFC₅₀0 of ginsenoside in stems and leaves was 127.63mg mL^-1,8.35 mg mL^-1,and 93.62 mg mL^-1 after 24-h,48-h,and 72-h treatments,respectively.However,the selective AFC₅₀0 of ginsenoside in stems and leaves was 72.87 mg mL^-1,17.23 mg mL^-1,and 77.10 mg mL^-1,respectively.The selective antifeeding activity was stronger than the non-selective antifeeding activity.The selective and non-selective anti-feeding rates of corn borers were higher with PDS（43.49%and 54.27%）than PTS（33.02%and 38.62%）.The selective and non-selective AFC₅₀0 values of PDS were 36.20 mg mL^-1 and 7.25 mg mL^-1 at the48^th h;however,the AFC₅₀0 values of PTS were 73.34 mg mL^-1 and 28.12 mg mL^-1 at the 48^th h,respectively.The non-selective AFC₅₀0 values of Monomer saponins Rb1,Rb2,Rc,Rd,Re,and Rg₁ were 0.18 mg mL^-1,0.04 mg mL^-1,0.37 mg mL^-1,0.08 mg mL^-1,1.24 mg mL^-1,and 0.87mg mL^-1 at the 48^th h,respectively.3.The ginsenoside in ginseng stems and leaves inhibited the population growth of Asiatic corn borers.The development duration of Asiatic corn borers was extended,and the survival rate of larva,average spawning number of adult female,oviposition days,and adult longevity were decreased with the increase of ginsenoside concentration.When the ginsenoside concentration of 40 mg g^-1 was used to treat corn borers,the total immature stage length of borer larvae was 6.02 d longer,the survival rate of larvae was 66.78%lower,the oviposition duration was 1.03 d shorter,the average spawning number of adult females was 166.7 granules less,the longevity of adult females was 0.84 d shorter,and the longevity of adult males was 1.04 d shorter than those of the untreated larvae control,respectively.The net reproductive rate,intrinsic increase rate,and finite increase rate of corn borers were 42.81%,25.00%,and 22.45%lower under the ginsenoside treatment than those of the control,but the mean generation time and population doubling time were 19.5%and 33.2%longer under ginsenoside than those of the control,when a high dose of ginsenoside at 40 mg g^-1 was used.4.The relative feeding rate,relative growth rate,approximate digestibility,food utility rate,and food conversion efficiency of Asiatic corn borers were all decreased with the increase of PDS concentration in the treatment,and they were reduced by 21.57%,11.74%,17.76%,25.80%,and 35.88%after the corn borers were fed with a high dose ginsenoside of 50 mg mL^-1compared with those of the untreated control,respectively.The types of amino acids,carbohydrates,and fatty acids in the larva were decreased after corn borers were fed with PDS.18 amino acids,5 carbohydrates,and 16 fatty acids were detected in the control group,while in the PDS treated group,L-valine acid,alanine,methionine,rhamnose,monolinolein,hexadecyl carbonate,or monoolein was even not detected.The relative contents of amino acids,monosaccharides and fatty acids were decreased with the increase of PDS concentration.The amino acid,carbohydrates,and fatty acid contents of the PDS treated group were 51.26%,73.69%and 54.75%lower than those of the control group.5.The activities of glutathione transferase（GTPS）,cytochrome enzyme（CYP450）,acetylcholin esterase（ACHE）,trehalase,phosphoesterase（ACP）,and alkaline phosphatase（ALP）in the 3th-instar corn borer larvae were inhibited by PDS.The activities of the above enzymes were reduced by 51.97%,34.73%,48.43%,46.42%,45.87%,and 39.55%when the3th-instar corn borer larvae were treated with 25 mg mL^-1 PDS for 72 h relative to those of the control group,respectively.The enzyme inhibiting rate was enhanced with the increase of PDS concentration at the same time,and it was also increased with the extension of treatment time at the same PDS concentration.The inhibiting rates on the above enzymes were 51.04%,33.35%,63.54%,85.50%,75.18%,and 42.66%higher after the 3th-instar corn borer larvae were fed with 25mg mL^-1 PDS for 72 h than those of the control without any feeding of PDS,respectively.6.Four groups of corn borer samples were analyzed with transcriptomics in order to conduct in-depth research in the antifeeding activity of ginseng stems and leaves.87.71 Giga Illumina sequence data were generated,and each sample had data of no less than 6.5 Giga.The annotated results were integrated and 31,698 unigenes were annotated.377,1119,and 1906differential genes were obtained at the PDS concentrations of 5 mg mL^-1,10 mg mL^-1,and25 mg mL^-1,respectively,relative to the control.118 GO term and 64 KEGG pathway enrichment results showed that there were 32 uigenes functioning in the metabolism pathway of fatty acids.The PPAR signaling pathway and fatty acid degradation pathway were significantly enriched by the PDS treatment over the control.These results revealed that the FATP and FABP genes in the PPAR signaling pathway were significantly down regulated.The above down regulation caused the expression of downstream genes SCD-1,ACBP,LPL,SCP-X,and ACO.Then the fatty acid degradation pathway was affected,so that,the interaction effect was produced.The FATP（c50620_g1 and c55114_g3）and FABP（c49424_g3）in the PPAR signaling pathway,and downstream SCD-1（c55056_g1,c48666_g1）and ACO（c56307_g1,c54537_g1,and c52770_g1）were verified through RT-qPCR.This result was identical as that from the transcriptome analysis.In addition,PDS decreased the enzyme activity through inhibiting the metabolism pathway of glutathione and catalyzing the expression of the related genes,such as GST,GPX,and CYP450.We concluded that the antifeeding activity of Asiatic corn borers may be caused by the reduction of the PPARs synthesis and fatty acid accumulation.In addition,PDS possessed significant inhibiting effect on the GSH metabolism pathway.PDS caused the confusion of nutrition metabolism and physiological function through inhibiting the activity of GST,CYP450,ACHE,trehalase,ACP,and ALP.The normal growthand development of corn borers were inhibited.This may be one reason for the antifeeding effect of ginsenoside in ginseng stems and leaves on Asiatic corn borers.This research elaborated the transcriptome of Asiatic corn borers fed with ginsenoside from ginseng stems and leaves,and clarified the molecular mechanism of the antifeeding activity.The dissertation research elucidated the effect of ginsenoside in ginseng stems and leaves on the biological activity of Asiatic corn borers and the ecological function of experimental population of Asiatic corn borers,analyzed the nutrition metabolism and physiological mechanism of Asiatic corn borers as affected by ginsenoside,and revealed the molecular mechanism of the antifeeding activity.This research provides the theoretical basis for exploitation of the stems and leaves of ginseng,reasonable utilization of ginseng resource,and application of ginseng stems and leaves in plant protection.This research is of high significance to understand the evolution pathway of ginseng,the mechanism of geoherbalism,and the pest control with ginsenoside related biological methods.