Cloning and Protein Expression of 8 Glycosyltransferase Genes in Group A of the Apple GT1 Family

MA Yingxin; SHANG Jinhai; YU Yishan; LIU Qian; ZHANG Xinying; JI Lusha; LI Pan

doi:10.19728/j.issn1672-6634.2024090001

Abstract：Glycosyltransferases(GT)are a class of enzymes that can glycosylated small molecule com-pounds,altering the biological functions of receptor molecules such as water solubility and stress resist-ance.To investigate the tissue expression pattern of candidate glycosyltransferase genes,qRT-PCR was used to detect the gene expression levels of 8 glycosyltransferase genes in the GT1 family group A in dif-ferent parts of apple plants.The results revealed that 7 glycosyltransferase genes,MdUGT91AJ1,MdUGT91AJ3,MdUGT91AJ4,MdUGT91AJ5,MdUGT91AJ6,MdUGT91AJ7 and MdUGT91C7 were expressed in different tissue sites with small variations,while MdUGT91AJ2 varied greatly.In order to heterologously express the proteins of candidate glycosyltransferases,these 8 genes were successfully cloned using apple'Gala'(Malus domestica Gala)cDNA as template,which were ligated into the prokary-otic expression vector pGEX-2T respectively,and the positive recombinant plasmids were transformed into E.coli(Escherichia coli)BL-21.After exploring the induction conditions,it was found that the optimal induction conditions for different glycosyltransferases are as follows:MdUGT91AJ1,MdUGT91AJ2,MdUGT91AJ3 are at 20 ℃,induced with 1.0 mmol·L-1 IPTG,and cultured for 16 h;MdUGT91AJ4,MdUGT91AJ5,MdUGT91AJ6,MdUGT91AJ7 were induced at 16 ℃ with 0.5 mmol·L-1 IPTG and cul-tured for 24 h;MdUGT91C7 was induced at 20 ℃ with 0.75 mmol·L-1 IPTG and cultured for 24 h.In conclusion,this study detected the gene expression levels of 8 glycosyltransferase genes in group A of GT1 family in different parts of apple plants,successfully constructed their prokaryotic expression vectors,and obtained their enzyme proteins after induced purification,which lays the foundation for the next step of glycosylation modification function identification of glycosyltransferases.