Artificial Cultivation And Gene Cloning And Expression Of Glycosyltransferase In Saponin Biosynthesis Of Anemone Flaccida

With continuous findings of the medicinal value of Anemone flaccida Fr.Schmidt,its wild resources have been excessively harvested in the past years,which results in sharp decline of its standing population.A.flaccida grows only in strict natural conditions with low propagation efficiency and slow growth rate,which restricts the exploitation and utilization of proprietary Chinese medicines.Therefore,it is of important significance to study appropriate cultivation techniques for guiding the artificial plantation of A,flaccida.In this dissertation,we investigated the temporal and spatial accumulative laws of triterpenoid saponin,an important medicinal component in A.flaccida,and conducted field experiments to study the effects of different cultivation techniques on plant growth and medicinal ingredients accumulation of A.flaccida.Moreover,based on the transcriptomic analysis,we cloned a glycosyltransferase gene that is involved in triterpenoid saponin biosynthetic pathway,and studied the temporal and spatial expression patterns of this gene in A.flaccida.The objectives of this study are to provide technological supports for large-scale cultivation and quality control of A.flaccida in the future.The main results are as follows:1.Total saponins content was significantly higher in rhizomes than in leaves with the highest content being found at withering stage.While,the contents of total saponins and W3 monomer in leaves were the highest at seedling stage.It suggested that the optimum harvest time for A.flaccida rhizomes is at withering stage.2.Seeding rhizomes with different lengths were planted and tested in field.The results showed that,the treatement of shorter seeding rhizomes had higher plant growth,rhizome production and total saponin and W3 monomer contents in rhizomes than the longer seeding rhizomes.In different plantation conditions,plant growth,rhizome production and the contents of total saponin and W3 monomer in rhizomes at harvest were the highest when plants grown under the coverage of one layer of sun-shading net and topdressing 75 kgN/ha at seeding stage.The fingerprint analysis showed that,there was no obvious difference in medicinal ingredients in rhizomes between artificially cultivated and wild plants.3.The transcriptome database of A.flaccida was established using rhizomes and leaves of wild plants as materials and Illumina Hiseq 2000 high-throughput sequencing platform as a tool.Based on the transcriptome data,two candidate glycosyltransferase genes,i.e.,85A1-GT and 27554-GT,which had a significantly higher expression in rhizomes than in leaves and might be involved in triterpenoid saponin biosynthetic pathway of A.flaccida,were screened out.The full length of coding sequence of these two genes was cloned using RT-PCR techniques and cloning vector pEASY-blunt.The recombinant pMetabV1-GT expression plasmids of the two genes were constructed and then transformed into Saccharomyces cerevisiae EBY100.The transformants were screened,cultured and their expression induced in the synthetic dropout medium.The SDS-PAGE analysis was used to detect the extracted and purified target proteins.The results showed that,the target protein expressed only by 85A1-GT gene was obtained.Enzymic determination by phenol red reagent method showed that,the protein had glycosyltransferase activity with 58U and specific activity of 4.64 U/mg.Therefore,85A1-GT was clarified as a glycosyltransferase gene and denominated as AfGT1.4.Based on the transcriptome data,10 candidate reference genes with their sequences were selected to study their temporal and spatial expression stability in A.flaccida.The results showed that,the combination of EF1A and ETIF1a can be used as reference genes to study the expression of target genes at different growth stages;while,the combination of PUBQ and ETIF1a can be used as reference genes in different organs at the same growth stage in A.flaccida.5.The temporal and spatial expression of glycosyltransferase gene AfGT1 was studied using EFTA and ETIF1a as reference genes.The results showed that,its expression was highest in rhizome at the flowering stage,but low in leaves at different stages and in rhizomes at other stages.The expression patterns of AfGT1 gene was generally in line with the temporal and spatial accumulations of total saponins,which suggested that AfGTl is an important gene involved in saponin biosynthesis in A.flaccida.