| Panax quinquefolius L.is a perennial herb of the genus Araliaceae.It is recognized as a nourishing,medicinal and edible plant in the world.It plays an important role in strengthening the body,improving eyesight,maintaining spirit,stopping convulsions,and prolonging life.2,3-oxidized squalene is the center of ginsenoside biosynthesis and is divided into the mevalonate pathway and the upstream pathway of 2-methyl-D-erythritol-4-phosphate(MEP)starting from pyruvate And downstream pathways for modification processes such as cyclization,hydroxylation,and glycosylation.The pharmacological activity of Panax species varies depending on the type and content of ginsenosides.Glycosylation is the final step in the synthesis of ginsenosides and a key step in the specificity and diversity of ginsenosides.In recent years,many scholars have done a lot of research on the glucosyltransferase gene synthesized by ginsenoside,but the research on ginseng-related glucosyltransferase is not enough.In this paper,the cDNA of the UDP-glucosyltransferase coding region was successfully obtained by RT-PCR using the laboratorypreserved callus of American ginseng as the basic material,and the three genes were ligated to the shuttle vector pAUR123.Then,it was transformed into Saccharomyces cerevisiae.After being disrupted by ultrasonic wave,the gene expression was detected by SDS-PAGE protein electrophoresis.The function of glycosyltransferase was determined by HPLC to detect the enzymatic reaction product.Finally,the fermentation temperature,time and rotation speed were three.The conditions are optimized and cultivated.The specific research results are as follows:1.Get the full length of cDNA coding sequence of three glycosyltransferases.The texture and growth of American ginseng callus was used as experimental material,and the total RNA of American ginseng was obtained by using the whole RNA extraction kit of Bao Bio.The cDNA of American ginseng was reverse transcribed by RTase-PCR using RT-PCR.The genomic PgUGT100,PgUGT101,PgUGT102 and PgUGT103 were used as templates to design the cloning primers.Three sequences were successfully cloned by using cDNA as template.After sequencing,the three sequences were determined to be 1428 bp,and 475 amino acids were translated.The encoded protein size was about 53 KDa.The sequence alignment and homology modeling of their amino acid sequences confirmed the target sequence and named the sequence.Sequence two and sequence three.2.Construct three heterologous expression vectors for glycosyltransferases.The glucosyltransferase genes sequence one,sequence two and sequence three were ligated with pAUR123 to construct pAUR123-sequence one,pAUR123-sequence two and pAUR123-sequence three three recombinant vectors,which are then transformed into INVSc1 Saccharomyces cerevisiae.In the process of colony PCR and double enzyme digestion,Y-pAUR123-sequence one,Y-pAUR123-sequence two and Y-pAUR123-sequence three were successfully constructed three yeast engineering bacterias.3.Analysis the expression of glycosyltransferases.The three engineering bacteria were fermented and cultured.The expression of the target protein in the engineered bacteria was detected by SDS-PAGE protein electrophoresis.The characteristic bands of the three target proteins in the protein electrophoresis map were 53 KDa.The results showed that the three sequences were The three Saccharomyces cerevisiae engineering bacteria were successfully expressed.4.Analysis the functional of glycosyltransferases.After the engineered bacteria are broken by ultrasonic waves,an enzymatic reaction is carried out.The enzymatic product was detected by HPLC,and it was confirmed that Pq-PPT-6,20-O-UGT can catalyze the conversion of PPT to F1 saponin,and catalyze the conversion of F1 to Rg1 saponin.It was confirmed that Pq-PPT-6-O-UGT can catalyze the conversion of PPT to Rh1.The catalytic activity of Pq-PPT-20-O-UGT was not detected.5.Optimal fermentation conditions of engineering bacteria.The optimized strains of engineering bacteria Y-pAUR123-Pq-PPT-6,20-O-UGT and Y-pAUR123-Pq-PPT-6-O-UGT were optimized from three aspects: fermentation temperature,fermentation time and rotation speed.The optimum fermentation temperature was 30?,the optimum fermentation time was 38 h,and the optimum fermentation speed was 270 rpm. |
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