Cloning And Prokaryotic Expression Of The Glycosyltransferase XynzUGT From Ligustrum Lucidum

Salidroside(chemical name:p-hydroxyphenethyl-?-D-glucoside,Salidroside)is a glueoside of tyrosol(chemical name:p-hydroxyphenylethanol).Salidroside is the most potent medicinal components of Rhodiola and first found in Rhodiola,hereafter,found in plants such as Ligustrum,Rhododendron,and Vaccinium.Pharmacological studies have shown that salidroside has good anti-fatigue,anti-anoxia,immunity enhancement,cardiovascular protection and other effects.Rhodiola and salidroside,are also used in many special environments to resist harsh environments,improve organism immunity,and have important application value in military,aerospace,sports,and health care,and also as Raw material medicines and additives are widely used in the industrial production of cosmetics,foods,health products,and medicines.In this study,the gene of xynzUGT was cloned through genetic engineering technology from Ligustrum quihoui Carr leaf,through transcriptome sequencing,eomparison with the reported glucosyltransferases that catalyze the synthesis of salidroside in Rhodiola rosea plants,The RACE used to clone xynzUGT.The gene was ligated respectively into the prokaryotic expression vectors pET-28a to construct recombinant vectors.Then the constructed recombinant vectors was transformed into E.coliBL21(DE3)respectively to build engineering bacteriasBL21-UGT-28a of prokaryotic expression.After culturing the engineering bacterias with IPTG inducing expression,verifying target protein through SDS-PAGE,Induction conditions optimization,solubility arnalysis,in order to lay the foundation for the subsequent study of enzymatic properties,The main contents of thisstudy are shown as follows:(1)The LigUstrum quihozui Carr leaves sprayed with tyrosol for 3 days were used as experimental materials,and the total RNA of Ligustrum quihoui Carr leaf was extracted for high-throughput sequencing.The RNA were transcribed by RT-PCR.According to the high-throughput sequencing results,RACE primer was designed to obtain the cDNA of 3'RACE,which was ligated with pMD19-T cloning vector.The constructed cloning vector was named pT-3'RACE.Transformed into E.coli DH5a,after bacterial liquid PCR identification,initially found to be constructed successfully,further sequencing to obtain a recombinant vector pT-3'RACE.(2)According to the sequencing results of pT-3'RACE,the 5'RACE primer was designed and the same method was used to obtain the recombinant vector pT-5'RACE.The results of two sequences were stitched together to obtain the full-length xynzUGT gene sequence,The design of specific primers in the cDNA as a template,finally get xynzUGT open reading fram.(3)The bioinfornatics of xynzUGT was studied.The molecular weight of the enzyme protein was 54,826.67 KD and the isoelectric point was 5.82.According to homology modeling and molecular docking studies,the results showed that the hydroxyl group and the side chain hydroxyl group on the phenyl ring of the tyrosol can interact with Glyl38 and Ser285 in enzyme proteins through hydrogen bonds.(4)The gene of xynzUGT was ligated into the cloning vector of pMD19-T to construct the reconbinant vector which is named pT-UGT.After that,transforming the recombinant vector into E.coli DH5a.Then performing bacterial fluid PCR and restriction enzyme digestion firstly,gene sequencing secondly,confirmed that the recombinant vector pT-UGT were successfully constructed.BamHI and xhol were used to digested the recombinant vector pT-UGT,the prokaryotic expression vector pET-28a was linearized by this two restriction enzymes respectively at the same time.Then the gene xynzUGT from vector pT-UGT was ligated into prokaryotic expression vectors pET-28a using T4 DNA Ligase to construct recombinant vector pET-28a?UGT.The recombinant vector pET-28a-UGT was then transformed into E.coli BL21(DE3)respectively.The engineering bacteria BL21-UGT-28a of prokaryotic expression were built as expected at last..(5)The built engineering bacteria was cultured respectively.After IPTG induced,the expression of the target protein from engineering bacteria was verified by SDS-PAGE.The result indicated that the target protein could be expressed as insoluble inclusion body in the engineering bacteria.The molecular weight of target band in BL21-UGT-28b was about 54.8KDa.Fernentation condition optimization for BL21-UGT-28a was preformed from three aspects including the concentration of IPTG,the inducing time,the inducing temperature.The optimal fermentation conditions of BL21-UGT-28a were the initial induced,the concentration of IPTG= 0.25mM,the inducing tinme 5 h,the inducing temperature 37?.(6)Soluble identification of the engineered bacteria revealed that it was expressed as an insoluble inclusion body.Tbree molecular chaperones,PG-KJE7,pG-KJE8,and pGro7,were used.The pG-KJE7 containing the dnaK-dnaJ-grpE chaperone had the best egg solubilizing effect,but the expression was extremely low.