Preliminary Study Of Polygonatum Cyrtonema Hua Adopting Molecular Pharmacognostic Approaches

Objective: To establish a rapid molecular identification method for Polygonatum cyrtonema Hua based on the full-length chloroplast genomes,to study and analyze the key enzyme genes(Pc UER1)in the synthesis of P.cyrtonema polysaccharides,that provided a theoretical basis for the further study of P.cyrtonema germplasm resources and the synthesis of P.cyrtonema polysaccharides and diosgenin.Methods:(1)The samples of P.cyrtonema were collected and DNA was extracted by improved CTAB method,which were sequenced,assembled and analyzed for its whole chloroplast genome.(2)Based on the sequence information of chloroplast genomes of P.cyrtonema,P.sibiricum and P.kingianum Coll.et Hemsl.,the sequences were aligned by clustal W in Bio Edit software.The specific primers of double blocking sites were designed to identify P.cyrtonema quickly.(3)RNA was extracted from the root,stem,leaf and rhizome of P.cyrtonema and screened the optimal products.RNA-Seq analysis and functional annotation were carried out,then speculating the biosynthesis pathway of polysaccharides and diosgenin in P.cyrtonema.(4)Based on the transcriptome sequencing data,the highly expressed enzyme gene Pc UER1 was screened for gene cloning,designing the specific primers Pc UER1-F and Pc UER1-R to conduct PCR amplification,and the band size was detected by agarose gel electrophoresis.After the bright DNA bands were cut from the gel and treated with agarose DNA gel recovery kit,the recombinant plasmid was constructed by p GM-T Fast cloning kit.The vector containing the target gene was introduced into E.coli DH5? competent cells by heat shock method and cultured.The cultured colonies were confirmed by colony PCR and cultured in LB(containing Ampicinin)medium,and the culture flora were confirmed the target gene again by colony PCR.The plasmid was extracted by small-scale plasmid extraction method,and the plasmid was sequenced after determining the plasmid concentration,and the target gene c DNA was analyzed by adopting to bioinformatics.The cloned gene Pc UER1,cloned from the c DNA of Pc UER1,that was recombined into the vector p ET-28a(+)containing Kanamycin by restriction site of 5' Bam HI and 3' Xho I to construct the recombinant plasmid Pc UER1 in p ET-28a(+).Results:(1)By sequencing the whole chloroplast genome of P.cyrtonema,the genome of 155 650 bp and the data of 7.04 Gb were obtained.The length of IR repeat region,long single copy region(LSC)and short single copy region(SSC)was respectively 25 526 bp,86 177 bp and 18 421 bp.Total number of 132(annotated by t RNAscan-SE)or 135(annotated by ARAGORN)chloroplast genes were annotated.(2)According to the fulllength sequence of whole chloroplast genomes of three sources of Polygonati Rhizoma,the specific variation sites of chloroplast gene sequence in 153 809 bp and 155 360 bp were found,and the bright bands of 1 507 bp were detected by PCY1-F and PCY1-R,conducting electrophoresis respectively according to the principle of block variation,which could effectively distinguish P.cyrtonema from P.sibiricum and P.kingianum Coll.et Hemsl..(3)The data of 42.03 Gb were obtained by transcriptome sequencing of roots,rhizomes,stems and leaves of P.cyrtonema.After assembling and removing redundancy,137 233 unigenes were obtained.According to the data of P.cyrtonema transcriptome,two biosynthesis pathways of P.cyrtonema polysaccharides and diosgenin were obtained.Among them,the key enzymes with number of 14 involved in the biosynthesis pathway of P.cyrtonema polysaccharides were ?-fructofuranosidase(INV),hexokinase(HK),UDP-glucose 4,6-dehydratase(RHM)and 3,5-epimerase/4-reductase(UER1),etc.The key enzymes involved in the diosgenin biosynthesis pathway were farnesy 1-diphosphate synthase(FDPS),farnesy 1-diphosphate farnesyltransferase(FDFT1),squalene monooxy genase(SQLE)and cycloartenol synthase(CAS1),etc.This study provided a reliable theoretical reference for the biological study of P.cyrtonema polysaccharides and diosgenin downstream.(4)The screened high expression enzyme gene Pc UER1 was cloned and the length of its c DNA sequence was 900 bp,encoding the number of amino acids was 299.Bioinformatics analysis showed that the secondary structure of amino acids was ?-helix,the second was randomcoil,and the ?-turn structure was the least.In the result of matching the protein with the template,the number of amino acids over 281 was matched successfully,accounting for 93.98 % of the total,indicating that the structure was consistent with the secondary structure.The recombinant plasmid DNA,providing the basis for the subsequent induction of recombinant protein expression and functional verification of the recombinant protein,that could also provide a reliable experimental basis for the functional verification of the recombinant protein and the expression of P.cyrtonema in various tissues.Conclusion:(1)Through the Illumina sequencing of the whole chloroplast genome of P.cyrtonema to obtain the full-length sequence of chloroplast gene,which laid a reliable data foundation for designing and screening specific primers.(2)A pair of specific primer sequences were designed and screened based on the chloroplast gene data,which could quickly identify P.cyrtonema from three sources of Polygonati Rhizoma and be of great significance for the effective identification of it.(3)The RNA extraction method for P.cyrtonema was optimized and the qualified RNA samples were used for subsequent RNASeq,to analyze the expression of genes in different tissues and annotate the gene function based on the transcriptome data.Speculating the synthesis pathway of polysaccharides and diosgenin in P.cyrtonema could provide reliable data for the screening of key enzyme genes of polysaccharide synthesis in downstream study and help for the subsequent experiment of diosgenin of P.cyrtonema.(4)Based on the transcriptome data of key enzyme gene expression in each polysaccharide synthesis pathway,the enzyme gene Pc UER1 was screened.After corresponding transformation and construction of recombinant vector,the expressed recombinant protein was obtained and expressed,which provided experimental basis and reference for proteomics research of P.cyrtonema.