Isolation,Identification And Functional Analysis Of Two UDP-glycosyltransferase Genes And Their Promoters In Panax Quinquefolius L.

Panax quinquefolous L.,also known as American ginseng,is one of world-recognized precious medicinal and edible plant.P.quinquefolous always used in anti-aging,enhance liver and regulation of immunity.The type and content of ginsenosides contained in P.quinquefolous are the two most important factors affecting the pharmacological activity of it.Starting from 2,3-oxidosqualene,the ginsenoside biosynthetic pathway can be divided into two major aspects: upstream of MVA pathway and MEP pathway,and downstream of cyclization,hydroxylation and glycosylation steps.As the final step in the ginsenoside synthesis pathway,glycosylation is an important step in determining monomeric saponin activity.Various glycosyltransferases catalyze glycosylation to modify the pharmacological and biological activity of ginsenosides in ginseng plants.Therefore,the current research hotspot is the glycosyltransferases in the ginsenoside synthesis pathway.In recent years,many studies have been conducted on glycosyltransferase genes in P.ginseng,but studies on glycosyltransferase genes in P.quinquefolous have not been reported.Promoter is located at the 5’ upstream regulatory region of the coding gene sequence and is responsible for the identification and integration of RNA polymerase.In the case of biotic or abiotic environmental factors stimulated,the cis-acting element on the promoter region of the coding gene regulates the downstream coding gene at transcriptional level by interacting with transcription factor or other trans-acting factors.Studies on the expression patterns of those promoters can help to further exploration of functional genes.In this study,We used P.quinquefolium callus as raw material to carry out gene cloning,recombinant vector construction,identification of heterogeneous expression,promoter cloning,genetic transformation and sequence analysis work of two glycosyltransferases.Based on the above research contents,the main research results are as follows:1.The full length of the cDNA coding sequence of two UDP-glycosyltransferase from P.quinquefolium were obtained by using smarter race technique.then,two UDP-glycosyltransferase were sequenced and named Pq3-O-UGT1 and Pq3-O-UGT2 respectively.We analyzed the amino acid sequence encoded by two UDP-glycosyltransferase gene and performed homology modeling.After submitting the encoding region cDNA sequence of two UDP-glycosyltransferase to GenBank,we get accession No.KR028477 and KR106207.2.After amplifying the cDNA sequence of two UDP-glycosyltransferase(Pq3-O-UGT1 and Pq3-O-UGT2),we constructed two recombinant yeast(Y-pAUR123-3GT1 and Y-pAUR123-3GT2)using enzyme digestion and ligation technology respectively.Enzymatic reaction products were analyzed by SDS-PAGE protein electrophoresis and HPLC-MS technology respectively.The results showed that the amino acids encoded by Pq3-O-UGT1 could catalyze the production of ginsenoside Rh2 from protopanaxadiol,and the amino acids encoded by Pq3-O-UGT2 could catalyze ginsenoside Rh2 and F2 to produce ginsenosides Rg3 and Rd.3.We amplified the gDNA sequence length of glycosyltransferase genes(Pq3-O-UGT1 and Pq3-O-UGT2)from the genomic DNA of P.quinquefolium.The result of sequence analysis showed that an intron structure with a size of 163 nt existed at the No.608 to 771 position of the Pq3-O-UGT1 gDNA sequence and an intron structure with a size of 12 nt existed at the No.152 to 164 position of the Pq3-O-UGT2 gDNA sequence.4.Using genome walking method,we cloned the 2611 bp length promoter sequence that located at 5’-upstream non-coding region of the Pq3-O-UGT1 gDNA sequence and the 1344 bp length promoter sequence that located at 5’-upstream non-coding region of the Pq3-O-UGT2 gDNA sequence from the genomic DNA of P.quinquefolium.The result of sequence analysis showed that there are a large number of cis-regulatory elements in these two promoter sequences.5.Six recombinant engineering bacteria were constructed by enzyme digestion and ligation technology and transformed into tobacco.The full-length promoter fragment and its 5’-deletions were merged with the β-glucuronidase(GUS)reporter gene and transferred into tobaccos to test their activities.GUS activity analysis showed that all six promoter fragments can drive GUS gene expression and have promoter activity.The results also showed that P-528 had the highest activity in four deletion fragments of the Pq3-O-UGT1 promoter,and its activity was 12.6 nmol MU/min/μg protein,which about 25% of the 35 S promoter activity.P-1420 has the highest activity in the two deletion fragments of the Pq3-O-UGT2 promoter,which is27.3 nmol MU/min/μg protein and about 64% of the 35 S promoter activity.The results of tobacco genetic transformation with 4 deletion fragments of Pq3-O-UGT1 promoter showed that low temperature and GA stimulation could significantly increased the activity of Pq3-O-UGT1 promoter full-length,while SA significantlyincreased the activity of deletion fragment P-1950.