Study On The Identification And Expression Of Functional Genes Related To Triterpenoid Saponins In Psammosilene Tunicoides

Psammosilene tunicoides W.C.Wu et C.Y.Wu is one of the Chinese traditional herbs,It is commonly used for the treatment of rheumatism,inflammation,traumatic injury.The main effective constituent of Psammoilene tunicoides is Oleanolic pentacyclic triterpenes total saponins,Such substances are mainly extracted from the root of Psammoilene tunicoides.It possessed the high pharmacological and economic value.For a long time,due to overdevelopment and slow growth,the wild resources and naturalyield of Psammosilene tunicoides has been reduced rapidly.It was not satisfied to the requirements for the drug.In this study,we obtained the key enzyme gene UGT(glycosyltransferase family gene).It was involving in the triterpenesaponins biosynthesis and building pYES2-?-AS reconstructed vectors for heterologous expression.This study will provide the direct production of active ingredients or intermediates to lay the foundation.The main research contents are as follows:1.According to the two of the UGT gene sequences of transcriptome of P.tunicoides,the ORF(Open reading frame)of cDNA sequences was obtained by PCR.Then TA cloning,sequencing,and sequence analysis were performed,they were named for UGT71G1 and PtT1 respectively.The UGT71G1 has a length of 1402 bp coding for 368 amino acids,which has a 1107 bp ORF.The protein molecular weight was 41.05 KD.The highly conserved functional domains of glycosyltransferase gene was found from 155 sites to 336 sites.The UGT71G1 was the most similar with Dianthus caryophyllus and Panax ginsengby NCBI blast.The 1529 bp sequence in P.Tunicoides named was PtT1 obtained,which has a 1377 bp ORF,encoding458 amino acids.The protein molecular weight was 51.25 KD.The PtT1 in P.Tunicoides was most similar with Dianthus caryophyllus DcT227 by NCBI blast.2.The UGT71G1?PtT1 and UGT1 were expression in prokaryotic expression system.The SDS-PAGE results of UGT71G1 and UGT1 showed that the expressed proteins were consistent with the anticipated size,and the stable prokaryotic expression system is established.The fluroescent quantitative PCR was used to detect the relative expression of the UGT1.It showed that the relative expression of the UGT1 was higher in the leaves was carried out.3.Additionally,heterologous expression vectors pYES2-?-AS were constructed through enzyme digestion and ligation technology respectively,then it transformed into Saccharomyces cerevisiae competent cell for heterologous expression identification.Collected the yeast extract and detected by bacteria liquid PCR,the results indicated that the genes could be expressed into protease with active function successfully.The UGT71G1 and PtT1 genes are successfully cloned,and the UGT71G1?UGT1 stable prokaryotic expression system is established,Heterologous expression vectors pYES2-?-AS were successfully constructed,this study will provide a foundation for the further purification,structural and functional researches of UGT protein.This study will provide the important basis for the study on the key role in the biosynthetic pathway of triterpenoid saponins of Psammosilene tunicoides and laying the foundation for the direct production of active compounds of Psammosilene tunicoides by Synthetic biology.