‘赤霞珠’葡萄果实中儿茶素没食子酰基化转移酶蛋白的纯化与鉴定  

Purification and identification of galloyltransferase involved in catechin metabolism in ‘Cabernet Sauvignon’ grape

作　　者：钱玉梅 李红侠[1] 李秀丽 吴萧 王晴 陈烨 高贵珍 QIAN Yumei;LI Hongxia;LI Xiuli;WU Xiao;WANG Qing;CHEN Ye;GAO Guizhen(School of Biological and Food Engineering,Suzhou University,Suzhou 234000,Anhui,China;Anhui Agricultural University/State Key Laboratory of Tea Plant Biology and Utilization,Hefei 230036,Anhui,China)

机构地区：[1]宿州学院生物与食品工程学院,安徽宿州234000 [2]安徽农业大学·茶树生物学及资源利用国家重点实验室,合肥230036

出　　处：《果树学报》2020年第8期1132-1143,共12页Journal of Fruit Science

基　　金：安徽省自然科学基金(1908085MC100);安徽高校自然科学研究重点项目(KJ2017A441);宿州学院教授(博士)科研启动基金项目(2016jb02;2016jb06);宿州学院自然科学重点项目(2017yzd11);宿州学院科研平台(2017ykf05);国家级大学生创新创业训练计划项目(201810379040)。

摘　　要：【目的】对参与‘赤霞珠’葡萄果实中催化1-O-β-D-没食子酰葡萄糖(1-O-β-D-glucogallin,βG)和表棓儿茶素(Epigallocatechin,EGC)生成表没食子儿茶素没食子酸酯(Epigallocatechin-3-gallate,EGCG)相关儿茶素没食子酰基化转移酶(epicatechin:1-O-galloyl-β-D-glucose O-galloyltransferase,ECGT)蛋白进行纯化与鉴定。【方法】以安徽萧县葡萄园种植的‘赤霞珠’葡萄果实为材料,采用丙酮沉淀、硫酸铵分段盐析、亲和柱层析、羟基磷灰石柱层析及Superdex200凝胶过滤色谱方法对ECGT酶蛋白进行分离与纯化,基于高效液相色谱(High Performance Liquid Chromatography,HPLC)技术测定以上各步纯化后获得的ECGT酶蛋白的比活力,并采用SDS-PAGE(SDS-Polyacrylamide Gelelectrophoresis,SDS-PAGE)电泳对Superdex200凝胶过滤色谱纯化后获得酶蛋白进一步分离与纯化,然后基于高效液相色谱-电喷雾质谱联用技术(HPLC-electrospray ionization mass spectrometry,HPLC-ESI-MS/MS)对目的条带(55 ku,28ku)进行鉴定;另外,进一步采用定量反转录-聚合酶链式反应(quantitative reverse transcriptase-polymerase chain reaction,qRT-PCR)技术对预测的ECGT酶蛋白相关基因及其上游3个糖苷转移酶基因(glucosyltransferase GT_(1-3))在果实不同发育时期的表达水平进行研究。【结果】以上蛋白纯化技术均能有效纯化ECGT酶蛋白,获得的ECGT酶蛋白比活力分别为0.32、3.14、11.78、31.80和88.82 U·mg^(-1),纯化倍数分别为9.81、36.81、99.38和277.56倍,酶活总回收率分别为83.33%、4.64%、0.96%和0.13%。对酶蛋白2条目的条带鉴定结果表明,丝氨酸羧肽酶-类酰基转移酶(serine carboxypeptidase-like acyltransferases,SCPL acyltransferases)家族中一条酶蛋白(XP_002272116.1)比对的分值均最高。此酶蛋白的相关基因SCPL及其上游相关3个基因GT_(1-3)表达均与‘赤霞珠’葡萄果实发育呈显著负相关。【结论】‘赤霞珠’葡萄果实存在ECGT酶蛋白,属于SCPL�【Objectives】The aim of this paper focuses on identification and purification of the galloyltransferase involved in catalyzing EGC andβG to generate EGCG in’Cabernet’grape,named ECGT.【Methods】The berries of’Cabernet Sauvignon’grape were picked from the Experimental Grape Garden of Xiao County for identifying ECGT and its gene expression.Firstly,the fine precipitated powder was collected by vacuum filtration based on acetone precipitation,then it was dissolved in Tris-HCl buffer for ECGT crude enzyme extraction after centrifuging at 12000 r·min-1 for 15 min at 4℃;ECGT protein was then purified by ammonium sulfate fraction,affinity column chromatography,hydroxyapatite chromatography,and Superdex 200 gel filtration chromatography based on its enzyme activity analyzed by HPLC.Secondly,the protein strips(55 ku,28 ku)were achieved by SDS-Polyacrylamide Gelelectrophoresis(SDS-PAGE)and identified by HPLC-ESI-MS/MS,and subsequently,the functional annotations of proteins were conducted by Mascot search engine(Matrix Science,London,UK;version 2.3.02)and Blast2GO program against the database of NR(the non-redundant protein database),KEGG(http://www.genome.jp/kegg/)and COG(http://www.ncbi.nlm.nih.gov/COG/).Thirdly,qRT-PCR was used to detect the expression level of genes including ECGT and 3 VvGT1-3 in fruits at different developmental stages.【Results】The specific activity of ECGT in the crude enzyme extraction was 0.32 U·mg^-1,and compared with crude enzymes,the specific activity was 3.14,11.78,31.80 and 88.82 U·mg^-1,respectively,via ammonium sulfate precipitation,ConA-affinity chromatography,hydroxyapatite chromatography and a Superdex 200 column.The purification-folds were 9.81-fold,36.81-fold,99.38-fold and 277.56-fold;the enzyme activity yields were 83.33%,4.64%,0.96%and 0.13%.According protein score,a protein SCPL acyltransferase(XP002272116.1)was the highest;the expressions of SCPL and 3 GT1-3 were all negatively correlated with fruit development.【Conclusion】The ECGT activity was detected i

关 键 词：‘赤霞珠’葡萄 SCPL类酰基化转移酶 纯化与鉴定 

分 类 号：S663.1[农业科学—果树学]