Screening And Cloning Of Sweet Orange UDP-Glycosyltransferase Genes And Exploration Of Its Prokaryotic Expression Conditions

Sweet orange(Citrus sinensis(L.)Osbeck)is an important economic fruit tree in China.It is a citrus plant in Rutaceae and contains rich glycoside compounds.Glycosylation modification is a widespread modification method in plants and plays an important role in the growth and development of plants.Glycosylation is usually carried out under the catalysis of UDP-glycosyltransferase(UGT),which is a process of transferring the glucose residue of UDP-glucose to the receptor molecule.UGT genes exist in the form of gene family in plants,and there are many members in this family.Therefore,it is difficult to clone the UGT genes when conducting plant glycosylation research.In this experiment,the young fruits of valencia(ovary,20 day after blowering,40 day after blowering)were used as the experimental material.Firstly,RNA from young fruits was extracted for transcriptome sequencing,and the sweet orange glycosyltransferase(Cs UGT)genes with high expression were screened.Secondly,we cloned the full-length c DNA of several Cs UGT genes and analyzed their coding proteins by bioinformatics.Finally,we constructed prokaryotic expression recombinant carrier and performed prokaryotic expression analysis of the successfully constructed recombinant protein,and explored its protein induced expression conditions.The Cs UGT genes screened in this study provide gene resources for subsequent gene function analysis.Exogenous expression of the protein encoded by Cs UGT genes also lays a certain foundation for subsequent studies on the biochemical function of UGT in vitro.The way to explore the inducible conditions of prokaryotic expression also provides some experimental ideas for future researchers to use prokaryotic expression for experimental analysis.The main results are as follows:1.Through transcriptome sequencing technology,we analyzed the overall data volume and data evaluation of the transcriptome,and assembled 7831 high expressed genes.Combined with the annotation information of the sweet orange genome database and motif identification,36 Cs UGT genes with high expression were screened.8 genes were randomly selected from 36 Cs UGT genes for q RT-PCR verification,and the verification results showed that the RNA-seq data were reliable to a certain degree.2.The proteins encoding 36 Cs UGT genes(Cs UGT)and 51 UGTs with known functions were selected to construct the protein evolutionary tree.Evolutional cluster analysis showed that 26 Cs UGTs could cluster with functional marker proteins.Among them,6 Cs UGTs were clustered with hormone glycosyltransferases,which means that they may be involved in the glycosylation modification of plant hormones,and 20 Cs UGTs were clustered with flavonoid glycosyltransferases,which means they may be involved in the glycosylation modification of flavonoids.3.The full-length c DNA sequences of 6 Cs UGT genes with complete ORF frames were successfully amplified by PCR.The c DNA sequence length of 6 Cs UGT genes were between 1383 to1512 bp.Bioinformatics analysis of 6 Cs UGTs showed that the amino acid number were between 460 to 503,the predicted molecular weight of the proteins between 51.70 to 56.55 k D,the isoelectric points between 5.44 to 6.69,the aliphatic index between 85.11 to 98.90,the number of non-polar amino acid residues(Asp + Glu)between 47 to 64,and the number of polar amino acid residues(Arg + Lys)between 39 to 59.The instability coefficients of 2 Cs UGTs were less than 40,which belonged to stable proteins,while the instability coefficients of 4 Cs UGTs were more than 40,which belonged to unstable proteins.Subcellular localization showed that 3 of the 6 Cs UGTs might be located in chloroplast and3 in cytoplasm.4.In this study,wo selected two prokaryotic expression vectors p Cold TF and p MAL-c5 X,and constructed eight recombinant plasmids successfully.8 recombinant plasmids were transfected into competent cells of Escherichia coli for prokaryotic expression analysis,and the conditions of protein induction were explored from the aspects of prokaryotic expression vector,prokaryotic expression host strain,protein induction temperature,induction time and IPTG inducer concentration.SDS-PAGE analysis showed that the eight recombinant proteins could express fusion proteins with the same molecular weight as the predicted proteins,but most of the fusion proteins existed in the form of inclusion bodies in the precipitation,and the content in the supernatant was less.The 8 fusion proteins mainly existed in Escherichia coli in the form of inclusion bodies.