Study On The The Main Chemical Contents Of 4-year-old Polygonatum Sibiricum And Clone The HMGS Gene

Polygonati Rhizoma is a tuberous rhizome of Polygonatum Mill.in Liliaceae.Polysaccharides,flavonoids,total phenols and saponins,which are the main active constituents of P.sibiricum,exhibit a broad range of pharmacological functions,such as regulating immunity,lowering blood sugar and anti-tumor.The market demand of P.sibiricum is increasing year by year because of it meets the regimen of modern people.However,due to excessive extraction of wild resources,the planting model of P.sibiricum has turned to artificial cultivation.Besidesthe accumulation of the main active constituents of P.sibiricum is affected by growth years,ecological environment,germplasm and cultivation methods.Therefore,it is of great significance to clarify the accumulation regularity of chemical components in P.sibiricum.In order to study the effect of cultivation modes on the accumulation of chemical components,we determined the chemical content of 4-year-old P.sibiricum from Lueyang,Shaanxi（field intercropping corn and imitating wild in the forest）;Optimized the extraction process of total saponins of P.sibiricum;Using the P.sibiricum transcriptome data to explore related enzymes of saponin biosynthesis pathway and analyze saponin synthesis pathway.This study provides a theoretical basis for the dynamic accumulation of the chemical components of the 4-year-old P.sibiricum and the regulation mechanism of saponins biosynthesis and metabolism.The main results are as follows:1.The dynamic changes of chemical contents in the 4-year-old P.sibiricum under two cultivation modes was studied.The results showed that the content of polysaccharide and flavonoid in rhizomeshave had similar changes,reaching the maximum content in August during the whole growth and development period of P.sibiricum.The content of polysaccharides and flavonoids in the rhizomes of P.sibiricum intercropped with maize were generally higher than that in the forest imitated wild P.sibiricum,while the flavonoid content in the leaves was lower than that in the forest imitated wild P.sibiricum.The content of total phenol was the highest in leaves under the two cultivation modes,showed a"V"changing trend,and the content in rhizomes was the lowest;Diosgenin in the rhizomes all showed a"M"-shaped change trend,with the highest content in July.From June to August,the content of diosgenin in the rhizomes of imitated wild P.sibiricumin the forestwas higher than that of the intercropped with corn.In this study,the accumulation regular patterns of main chemical components in P.sibiricum had been clarified.Except for June,the content of polysaccharides in the two cultivation modes reached the requirements of the Pharmacopoeia.Therefore,both cultivation modes can be adopted.2.Study on ultrasonic extraction technology for total saponins of P.sibiricum.Based on the single factor test,the ethanol concentration,material-to-liquid ratio,ultrasonic power,extraction time and extraction times,the best process parameters for total saponins extraction were obtained by orthogonal experiments.The optimal technological conditions for ultrasonic extraction of total saponins were as follows:70%ethanol concentration,1:15 ratio of material to liquid,and ultrasonic power of 500W,ultrasonic extraction 3 times,each extraction time was 30 min.The extracted content was 21.66 mg·g^-1,which was 0.95 mg·g^-1 lower than the best single factor process,but there was no significant difference.However,the optimized extraction process could reduce the extraction cost from materials（1:20 to 1:15）and time（100 minutes to 90 minutes）.3.The total saponins content in the rhizomes of Polygonatum has been determined,and there was a significant difference between 1 and 2-year-old rhizome.Therefore,transcriptome sequencing was performed on them,a total of 80574126～91933624 Clean reads were obtained.After de novo transcriptome assembly,435858 Unigenes with an average length of 1085 bp were obtained,of which 264547（60.69%）can be annotated in any of NR,NT,KO,Swiss Prot,PFAM,GO,KOG and 29,548（6.77%）can be annotated by the 7 major databases at the same time.KEGG metabolic pathway analysis of P.sibiricum found that a total of 10718 Unigenes were annotated to the synthesis of secondary metabolism,involving 131 KEGG metabolic pathways.Among them,965 Unigenes were annotated to 3 saponins biosynthetic stages including Terpenoid backbone biosynthesis,Sesquiterpenoid and triterpenoid biosynthesis,and Steroid biosynthesis.Based on statistical analysis of differentially expressed genes in the 1and2-year-old rhizome,15 enzymes such as ACAT,HMGS,and DXS were identified in the above 3metabolic stages,which were encoded by 75 Unigenes,of which 24 Unigenes were significantly different.4.In this study,HMGS1 and HMGS2 gene were cloned from P.sibiricum by RT-PCR.The sequence alignment results showed that the similarity of HMGS1 and HMGS2 sequences was93.71%,and they had high homology with Asparagus officinalis.The phylogenetic tree results show that the HMGS1、HMGS2 and A.officinalis with Narcissus tazetta in the same group,indicating that their HMGS has high homology,and it is speculated that they have a common evolutionary ancestor.The results of the relative expression levels of HMGS1 and HMGS2 and the total saponin content in different tissues of P.sibiricum showed that the HMGS expression in the aerial part was not synergistic with saponin content.However,in underground rhizomes,there was a certain synergistic relationship between the expression level of HMGS and the content of saponin.The following reasons were inferred:（1）The saponins were synthesized and accumulated at different locations.Although saponins were synthesized in the stems,they were not accumulated and stored.After the saponins are synthesized,they were transported to fruits and rhizomes for storage.（2）The high expression of HMGS gene in the stem may be related to the self-protection of the cell.The HMGSs can increase the content of sterols to maintain cell morphology and membrane registration.Further study of the stress effect of methyl jasmonate（Me JA）on HMGS of P.sibiricum seedlings,it was found that the expression of HMGS1 and HMGS2 genes increased first and then decreased with the increase of Me JA treatment time.The expression of both reached the maximumat 24 H.And the results showed that HMGS gene can be induced by Me JA,and the optimal time for Me JA to induce stress on P.sibiricum seedlings was 24 H.