Gene Cloning And Recombinant Expression Of Rutin Degrading Enzyme In Fagopyrum Tataricum

Rutin,which is a product of secondary metabolism in tartary buckwheat(Fagopyrum tataricum),has been widely used in the clinic because its antihyperglycaemic,antihypertensive,and antioxidative properties.Rutin can be degraded easily by rutin degrading enzyme(RDE),so it’s necessary to clone the gene of RDE for the study of rutin metabolism.There are many studies on the extraction and characteristic determination of RDE but no research on the gene cloning.Based on the tartary buckwheat floral transcriptome assembly(GeneBank accession No.SRX025322)previously reported,the following work was done.The N-terminal amino acid sequence was determined by automated Edman degradation and tanderm mass spectrometry(MS)was used to obtain 15 inner amino acid sequences.Then the 16 peptide sequences were searched against SRR063703 through tblastn and ESTs of RDE was obtained.Primers were designed for the amplification of RDE gene segments.Amplicons by different pairs of primers were sequenced,the assembly of several correct sequences created a 1431 bp complete coding sequence(cds)of mature RDE.Blastn result showed no similar sequence was searched in the database,which implied it’s a novel sequence.Deduced Amino acid sequence was aligned by blastp and the highest similarity was only 62%with β-glucosidase in Polygonum tinctorium(GeneBank accession BAA78708.1).The result explained the specificity of RDE as a special β-glucosidase.Secondary structure prediction result demonstrated a high ratio of random coil(40.67%).The cds of mature RDE was cloned into pET-2 8a(+)and overexpressed in E.coli Rosetta gami2(DE3).After renaturation of inclusion bodies and cobalt affinity chromatography,the enzymatic activity was determined.There was no clue of catalytic activity possibly due to a loss of glycosyl groups compared to natural mature RDE.Western blot result showed the specific binding to natural RDE antibodies of recombinant RDE.In this work,the gene of RDE was firstly cloned successfully and further confirmed by Western blot.The method of cloning can be used for other unknown sequences.The sequence of RDE lays the foundation for the studies of rutin metabolism.RDE can also be applied to the field of nonaqueous enzymology because of its pH and organic solvent tolerance.