Cloning And Expression Of ?-d-glucosidase Gene In Phlorizin Degradation Pathway

Phenolic acids are widely distributed in nature and are secondary metabolites closely related to the growth of higher plants.More and more studies have shown that phenolic acids play an important role in allelochemicals.As one of the most phenolic compounds in apple plants and continuous cropping soil,phlorizin plays an important role in the storage of phenolic acids.At the same time,microbial activity is the main way to degrade phenolic acids in soil.Therefore,the study of microbial degradation of phenolic acids has become a hot issue.In this study,Novosphingobium subarcticum AMCC100102 which was isolated and preserved early in the laboratory was used as the experimental object,the degradation products of phlorizin were analyzed by HPLC and the degradation pathway was deduced.The primary study gene which coding?-D-Glucosidase,was identified on the basis of the degradation pathway.Transposon mutagenesis was used to screen the mutants in the early experiments,but no results were obtained.Then,the genome of AMCC100102 was sequenced and found that it contained 11?-D-Glucosidase genes.Differential expression of these 11genes were detected by differential culture of AMCC100102,two differential genes were detected and had carried on the exogenous expression of these two genes.The main results were as follows:?1?The AMCC100102 was cultured with phlorizin as the sole carbon source and the degradation products in the fermentation broth were detected by HPLC.A total of six phenolic compounds were detected and they were phloretin,phloroglucinol,p-hydroxyphenylpropionic acid,p-hydroxyacetophenone,p-hydroxybenzoic acid and protocatechuic acid.The degradation pathway of phloridzine was deduced?Fig.3-3?.?2?The sequence of the genome of AMCC100102 was determined by Illumina sequencing.The results showed that the genome size was about 6.11MB and the GC content was 65.73%.There were 5974 genes with an average length of 938bp.The genome of AMCC100102 had the highest similarity to the genome of Novosphingobium resinovorum and Sphingomonas,reaching 47.87%and 20.39%,respectively.?3?By comparing the genomic data of AMCC100102 with GO,KEGG,COG,NR database,11?-D-Glucosidase genes were found.AMCC100102 was cultured with glucose and phlorizin respectively.AMCC100102 was cultured with glucose and phlorizin as the sole carbon source,respectively.The expression of these 11 genes was detected by reverse transcription PCR,and only bdg5 and bdg6 were differentially expressed.bdg5 is expressed only in the treatment of phlorizin as a carbon source;bdg6 is expressed in the treatment of glucose and phloridzin,but the expression level in the treatment of phlorizin is greater than that in glucose treatment.By conservative domain analysis,BDG5 belongs to the BgIB superfamily and BDG6 belongs to the Glyco-hydro-1 superfamily.?4?The prokaryotic expression vectors of bdg5 and bdg6 were constructed and named pET30a?+?-bdg5 and pET30a?+?-bdg6,respectively.The results showed that the fusion proteins BDG5 and BDG6 had the activity of phlorizin degradation by silica gel plate GF₂₅₄thin layer chromatography.The purified fusion protein was purified by Ni-NTA purification.