苹果GT1家族A组中8个糖基转移酶基因克隆及蛋白表达  

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

作　　者：马迎新 商金海 于薏珊 刘倩[1] 张新莹 冀芦沙[1] 李攀 MA Yingxin;SHANG Jinhai;YU Yishan;LIU Qian;ZHANG Xinying;JI Lusha;LI Pan(School of Pharmaceutical Sciences and Food Engineering,Liaocheng University,Liaocheng 252059,China)

机构地区：[1]聊城大学药学与食品工程学院,山东聊城252059

出　　处：《聊城大学学报(自然科学版)》2025年第2期307-316,I0001-I0005,共15页Journal of Liaocheng University:Natural Science Edition

基　　金：国家自然科学基金项目(32001982);山东省自然科学基金项目(ZR2021MC062)资助。

摘　　要：糖基转移酶(Glycosyltransferase,GT)是一类可糖基化修饰小分子化合物的酶类,可改变受体分子的水溶性、抗逆性等生物学功能。为探究候选糖基转移酶基因的组织表达模式,qRT-PCR检测GT1家族A组中8个糖基转移酶基因在苹果植株不同部位的基因表达水平。结果发现,7个糖基转移酶基因MdUGT91AJ1、MdUGT91AJ3、MdUGT91AJ4、MdUGT91AJ5、MdUGT91AJ6、MdUGT91AJ7和MdUGT91C7在不同组织部位中均有表达,差异较小,而MdUGT91AJ2差异较大。为了异源表达候选糖基转移酶的蛋白,以苹果‘嘎拉’(Malus domestica Gala)cDNA为模板成功克隆了这8个基因,分别将其连接到原核表达载体pGEX-2T中,将阳性重组质粒转化到大肠杆菌(Escherichia coli)BL-21中。经过诱导条件探索,发现不同糖基转移酶的最佳诱导条件如下:MdUGT91AJ1、MdUGT91AJ2、MdUGT91AJ3为20℃,1.0 mmol·L^(-1) IPTG诱导,培养16 h;MdUGT91AJ4、MdUGT91AJ5、MdUGT91AJ6、MdUGT91AJ7为16℃,0.5 mmol·L^(-1) IPTG诱导,培养24 h;糖基转移酶MdUGT91C7为20℃,0.75 mmol·L^(-1) IPTG诱导,培养24 h。总之,本研究检测了GT1家族A组中8个糖基转移酶基因在苹果植株不同部位的基因表达水平,成功构建了其原核表达载体,经诱导纯化后获得了它们的酶蛋白,为下一步糖基转移酶糖基化修饰功能鉴定奠定了基础。Glycosyltransferases(GT)are a class of enzymes that can glycosylated small molecule compounds,altering the biological functions of receptor molecules such as water solubility and stress resistance.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 different 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 prokaryotic 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 cultured 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.

关 键 词：苹果 糖基转移酶 基因克隆 蛋白表达 

分 类 号：S661.1[农业科学—果树学] Q943.2[农业科学—园艺学]