Studies On A New Glycosyltransferase In Trigonella Foenum-graecum L

Fenugreek (Trigonella foenum-graecum L) is a kind of annual herb Its dried ripe seeds have the function of warming the kidney, resisting coldness and easing pains,which is used for the treatment of the coldness caused by deficiency in the kidney, the coldness and pains in the lower abdomen, the hernia in the intestinal and the alpine athlete’s foot, thus having been included the People’s Republic of China Pharmacopoeia.Glycosyltransferases (GT) is a kind of proteins with special biological activities, which widely exist in the prokaryotes, eukaryotes, archaea, and viruses. They can transfer the glycosyl donor with biological activities to the certain receptor molecules, while their receptor molecules can be saponins, hormones, proteins, etc.in this study,Fenugreek, alfalfa (Medicago FJ477891.1), licorice (Glycyrrhiza echinata AB098614.1) and Lotus corniculatus(Lotus japonicus AK337125.1) belong to the same genus,fenugreek was compared with the other three kinds of plants on homology. According to the obtained comparison outcome, a pair of degenerate primers(upper primer:F1:F1:5’-C A A T T C T A C (T/G) A A T (C/A) G A G A A-3’; lower primer:R1:5’-A C A (G/C) G C C (T/G/C) T T C T C T A T-3’) were got and used for the action of PCR, obtaining a piece of conserved sequence of128bp. Then4pieces of specific primers(F2:5’-G C T T G T A A C T G A T G T G C G A A G-3’,F3:5’-G T A A C T G A T G T G C G A A G G A T-31’, R2:5’-C A C C G A C A C T C A C T C C A A T C-3’, R3:5’-G A C A C T C A C T C C A A T C C T T C-3’) were designed for the conserved sequenced, and were used for3’RACE and5’RACE. After sequenced,3’RACE and5’RACE products were spliced, getting the whole CDS sequence of the gene. The gene, whose sequence length is1455bp, codes a piece of polypeptide, whose relative molecular mass is54.204kD. The polypeptide consists of484amino acids and its isoelectric point is5.16. In this study, the gene was named after GT3to be described conviniently. The analysis of conserved domain in the NCBI and Prosite databases indicated that GT3has the concerved domain of UDP-glucose glycosyltransferase, a GTB-type glycosyltransferase superfamily, and that it is a kind of glycosyltransferase gene. The analysis of membrane-spanning domain in the Split and TMPred databases indicated that the enzyme coded by GT3may have a membrane-spanning domain from140to160.In the further research of this study, another piece of special primer(F4:5’-C G C G G A G C T C A T G G A A G G T G T T G A A C G A C C-3’,R4:5’-C G C G G C G G C C G C T T A A T C A A G T G G T T T T G G G A T A T C T-3’) was designed to amplificate the entire sequence via PCR with the total mRNA from fenugreek seedlings used as templates. Then the amplificated products were sequenced and transferred into the expression vector pET-28a to get the prokaryotic expression vector pET-28a-GT3, which was finally transformed into competent E. coli BL21(DE3). After IPTG induction and SDS-PAGE detection, the wanted protein strip was successfully detected and the protein existed in the sediment, which led the preliminary conclusion that the protein is a kind of membrane protein.In the study, a new glycosyltransferase was successfully obtained from the fenugreek, which is very important for deep researches on the enzymes,especially on the glycosyltransferases, in the fenugreek. In the following researches, the glycosyltransferase’s biological activities will be studied further.