| Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.is an important perennial herb under the forest belonged to Paris in Trilliaceae,and it is an important raw material for large pharmaceutical companies such as Yunnan Baiyao Company.The wild resources are currently listed as endangered natural drugs due to excessive excavation in Yunnan province.There are some problems such as mixed sources,mixed quality and the slow growth rate are far from meeting the needs of the current pharmaceutical market.Therefore,the excellent genes related to the synthetic pathway of saponins from Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.will be tapped to further explore the molecular regulation mechanism of saponin synthesis in Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.to improve the saponin content through gene regulation,It is of great significance in raising economic efficiency and ecological benefits such as improving the industrial efficiency of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.,protecting the wild resources of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.,increasing the income from agriculture and forestry in mountain areas and promoting the benign circulation of the agroforestry ecosystem.In this study,eight geographical provenances of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.in Jingdong of Pu'er,Chengjiang of Yuxi,Jianchuan of Dali,Yongsheng of Lijiang,Dayao of Chuxiong,Qiubei of Wenshan,Malong of Qujing and Zhenxiong of Zhaotong were collected in Yunnan province,and the source of Jingdong provenance with higher growth rate and higher total saponin content was selected as research object.The saponin differences in Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.of different tissue or organ and different geographical provenances were studied in different seasons by determining the content of saponins of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz..The transcriptomes were sequenced in different parts of the plant in different seasons when the content of saponins was highest or lowest,and some candidate genes related to saponin synthesis were screened out.Twenty of these genes were tested by real-time quantitative PCR and then correlated with saponin content.The purpose is to discover the superior genes in the saponin synthesis pathway of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.and further explore the bioregulatory mechanism of saponin synthesis in Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz,and provide scientific basis for increasing the effective saponin content in medicinal plants.The results showed:(1)Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.from Jingdong provenance had the characteristics of long growth period as long as 10 months in a year,high content of saponins of 1.57%,and fast growth rate of 17.76%.It can be used as a superior source with high-quality for replanting.(2)Saponins were detected in rhizomes of 1-5 years,leaves,fibrous roots,stems,leaves,flowers,buds,pericarp,seeds and other parts from Jingdong provenance of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz..The contents were ranked from high to low: rhizome> leaf> seed> buds> stems> fibrous roots> pericarp> flowers.Although the content of saponins in the other parts except the rhizomes did not exceed 0.6%,it still had the value of development and utilization,especially in the stems and leaves,and the content was about 0.16-0.52%.The content of rhizomes from 1-5 years was ranked in descending order: biennial rhizomes> three-year rhizomes> four-year-old,five-year rhizomes> annual rhizomes,of which two-year-old part had the highest total saponin content and annual ones had the lowest.(3)The annual cycle of saponin content in the Jingdong provenance was as follows: The content of saponin was highest around the dormant period in January,while the content of saponin was lowest in the flower and fruit period,and it was moderate in the fast growing period of the rootstock.(4)A total of 69.84 Gb Clean Data were obtained by sequencing the transcriptomes of 8 samples of Jingdong Provenance.The Clean Data all reached 6.15 Gb,and the percentage of Q30 base was 85.63% or more.A total of 79,867 Unigenes were assembled and the N50 of Unigene was 1,361,and the assembly integrity was high.41,253 annotations were obtained for Unigene and 14,769 differential genes were obtained in KEGG.The transcriptome data of 8 samples were compared and analyzed.There were 23,816 differential genes,among which 13,183 Unigene were up-regulated and 10,633 Unigene were down-regulated.(5)By analyzing the functional annotation data of the transcriptome gene in the transcriptome,the genes involved in the synthesis of saponins were screened and involved the upper,middle,and downstream pathways of steroidal saponin synthesis respectively.In the MVA pathway,a total of 6 synthetases involved 30 unigenes;in the MEP pathway,annotated to 7 synthetases involved 10 unigenes;a series of candidate genes involved in steroidal skeleton synthesis involving 6 synthetases and 18 Unigenes including steroidal skeletonbone biosynthesis and downstream pathway of steroidal sapogenin oxidation and modification process,in which 221 Unigenes involved in 3 types of synthetase;272 Unigenes involved in 22 metabolic pathways in secondary metabolic pathways related to steroidal saponin biosynthesis were excavated as follows: AACT[EC:2.3.1.9]??-AS[EC:5.4.99.39]?CAS[EC:5.4.99.8]?SE [EC:1.14.99.7] ? SQS[EC:2.5.1.21] ? FPPS[EC:2.5.1.10] ? GPPS[EC:2.5.1.29] ?IPPI[EC:5.3.3.2] ? HDR[EC:1.17.1.2] ? HDS[EC:1.17.7.1] ? MDS[EC:4.6.1.12] ?CMK[EC2.7.1.148] ? MCT[EC:2.7.7.60] ? DXR[EC:1.1.1.267] ? DXS[EC:2.2.1.7] ?PMD[EC:4.1.1.33] ? PMK[EC:2.7.4.2] ? MVK[EC:2.7.1.36] ? HMGR[EC:1.1.1.34] ?HMGS[EC:2.3.3.10]?UCTs[EC:2.4.1.173]?CYPs[EC:1.14.13.21] etc.It covered almost all the nodes in the steroidal saponin synthesis pathway,enriching the information on enzyme genes of the saponin carrier in biosynthetic pathway of steroidal saponins,and helping us to better understand saponin synthesis pathway.(6)Through the comparison of the differential genes in each sample of the Ko00100 pathway synthesized by saponin,it was found that the following genes in the steroid synthesis pathway were differentially expressed among the tissue samples: FDFT1?ERG6?SMT1?SMT2?SMO2?DHCR24?DWF1?HYD1?TAG?SMO1?ERG3?STE1?SQLE,ERG?CYP51G1?CYP51?SC5DL?Delta7-sterol5-desaturase,squalene monooxygenase,cholesterol ?-isomerase,sterol 14-demethylase.These genes are involved in the metabolism of saponins and they are important candidate genes for saponin synthesis.(7)In the saponin metabolic pathway,20 differential genes were selected from the above genes,and q PCR was performed on samples from different tissue sites,different growth seasons,and different provenances.The situation is basically positively related to its saponin content,There was a positive correlation between expression levels and saponin content in different geographical provenances;17 genes expression levels in 20 genes in different tissues or organs were positively correlated with saponin content in different tissue or organ;8 out of 10 genes in rhizomes in different growing seasons.which can be preliminarily inferred that the above genes may be involved in the synthesis of saponins.(8)The full-length sequence of the HMGR(3-hydroxy-3-methylglutaryl coenzyme A reductase)gene was cloned from the rhizome of Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.,and the full-length 1494 bp ORF was obtained,encoding 497 amino acids and designated Pp HMGR(Gen Bank Accession No: MG601751);Bioinformatics method was used to predict and analyze the physicochemical properties,protein structure and function of HMGR protein.Similarity search was performed on the protein and the evolutionary tree was constructed.The amino acid sequence was found to be conserved in HMGR protein.The sequence had the highest similarity with Fritillaria cirrhosa(Fu/ASO 66849.1),which reached 83%.The expression of the gene in the different issues was observed by using real-time fluorescence quantification method.The results showed that the expression of Pp HMGR in rhizomes was significant.Above the leaves,stems and shoots on the ground,the expression of rhizomes for 3 years was significantly higher than that of 1 or 5 years. |
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