Screening, Cloning And Expression Of 6 Wild Licorice Endophyte-specific Glycosyltransferases

Endophytic fungi are closely related to medicinal plants and can directly or indirectly participate in the growth,development and accumulation of pharmacodynamic components of medicinal materials in a variety of ways.Glycosyltransferases(GTs)are enzymes that are the last step in the biosynthesis of glycosides.With the development of sequencing technology,glycosyltransferase family genes have been found in the genomes of endophytic fungi of medicinal plants,and genes of the same family may have the same or similar function.Endophytic fungi have the potential to affect the quality formation of traditional Chinese medicinal materials,which may be related to genes of the same family as the two.However,no studies have been reported on the relationship between genes from the same family of the two.Therefore,in this study,we established a species library of endophytic fungi from wild Glycyrrhiza uralensis,and performed whole genome sequencing of endophytic fungi from six different species of wild Glycyrrhiza uralensis,and found that endophytic fungi have specific glycosyltransferases closely related to plant glycosyltransferases,and successfully obtained their soluble proteins,laying the foundation for studying the catalytic activity of specific glycosyltransferases from endophytic fungi and helping to elucidate the potential effects of genes of the same family of endophytic fungi and medicinal plants on the biosynthesis of pharmacodynamic active components at the molecular level.The main research contents and results of this paper are as follows:1.Establishment of endophytic fungal species library of wild Glycyrrhiza uralensis and study on the species diversity of endophytic fungiWild Glycyrrhiza uralensis was collected from Jirangtu Town,Hangjin Banner,Ordos City,Inner Mongolia.In this study,endophytic fungi were isolated by tissue block isolation,and a total of 214 endophytic fungi were isolated and purified from 220 tissue blocks of wild Glycyrrhiza uralensis,and the species were comprehensively identified using morphological,microscopic,and molecular biological techniques,which were assigned to 30 endophytic fungi belonging to 4 classes,5 orders,6 families,9 genera,with an isolation rate(IR)of 97.27%and a colonization rate(CR)of 97.27%;the dominant genus of endophytic fungi in wild Glycyrrhiza uralensis was endophytic fungi of Fusarium,with a relative frequency(RF)of 63.08%,followed by endophytic fungi of Aspergillus and Alternaria,with relative frequencies of 11.68%and 8.41%,respectively.2.Whole genome sequencing and bioinformatics analysis of six wild Glycyrrhiza uralensis endophytic fungiThe whole genome sequencing of endophytic fungi from six wild Glycyrrhiza uralensis strains of different species was completed using the Illumina PE150 sequencing platform,and the strains were numbered C3,LB,Y3,Y4,Y26,and F9 at a sequencing depth of 100 ×.The sizes of the assembled genomes were 58,37 Mb,44.51 Mb,57.10 Mb,36.52 Mb,109.75 Mb,and 78.27 Mb,respectively;2601,290,2539,194,3932,and 1210 contigs larger than 500 bp were obtained after assembly;the GC contents of the assembled genomes were 51.55%,48.86%,48.15%,52.89%,52.27%,and 51.63%,respectively;the numbers of predicted genes were 9790,8676,9197,7532,15236,and 12500,respectively;the numbers of genes annotated to the GO database were 6708,6000,63 84,5193,10996,and 8651,respectively;the orders of the numbers of genes annotated to the KEGG database were 9200,8222,8795,6390,14252,and 10891,respectively;the number of genes annotated to the NR database was 9215,8323,8900,6607,11009,and 9532,respectively;the biosynthetic gene clusters of potential natural products were.27,35,33,19,40,and 34,respectively.Based on the results of bioinformatics analysis,it was found that endophytic fungi had a large number of genes of the same family as plants,which laid a foundation for subsequent study on the relationship between the genes of the same family as the two.3.Screening,cloning and expression of specific glycosyltransferases from endophytic fungiIn order to study the relationship between endophytic fungi and glycosyltransferases of the same family of plants,in this study,the complete genome sequences of 6 endophytic fungi were compared with the sequences of 91 plant glycosyltransferases,and a total of 15 endophytic fungi with high match to plant UGTs were obtained;then 15 candidate genes were analyzed for phylogenetic analysis and conserved motif alignment with 91 plant UGTs,and it was found that 4 of the candidate genes were closely related,and had partially conserved motifs similar to plant UGTs,so they were used as specific glycosyltransferase genes of endophytic fungi for the next step.The four specific genes were from strains Y3,Y26,C3,and LB,which were named Y3EFGT(genome ID:A3812),Y26EFGT(genome ID:A07288),C3EFGT(genome ID:A8031),and LBEFGT(genome ID:A7482),respectively.Total RNA of endophytic fungi Y3,Y26,C3 and LB was extracted,cDNA was obtained by reverse transcription,primer sequences of the target gene were designed,and bands consistent with the length of the target gene were cloned.Sequencing results showed that the similarity with the target gene sequence was 100%,and the sequence lengths of Y3EFGT,Y26EFGT,C3EFGT and LBEFGT genes were 1626 bp,1473 bp,1473 bp and 1614 bp,respectively.Bioinformatic analysis of the specific genes of the four endophytic fungi showed that the similarity of the tertiary structure of glycosyltransferase proteins of plant or microbial origin with verified catalytic function of the four specific genes was high,indicating that the specific genes have the potential to participate in similar physiological and biochemical reactions.Four specific genes were ligated into the prokaryotic expression vector pEASY-Blunt E1,and the recombinant plasmids Y26EFGT-pEASY-Blunt E1,Y3EFGT-pEASY-Blunt E1,C3EFGT-pEASY-Blunt E1,and LBEFGT-pEASY-Blunt E1 were successfully constructed;the recombinant plasmids were transformed into E.coli BL21(DE3)competent cells to induce the expression of the target protein,and the soluble Y26EFGT recombinant protein was successfully obtained by SDS-PAGE electrophoresis.