荔枝果皮BPox的分离纯化及其在果实成熟过程中的表达  

Purification,Characterization and Expression of Ionically Bound Peroxidase in Litchi Pericarp during Coloration and Maturation of Fruit

作　　者：郭志雄[1,2] 孙冷雪 郑嘉敏[2] 蔡灿军 王蓓[2] 李开拓[2] 潘腾飞[1,2] 佘文琴[1,2] 陈桂信[1,2] 潘东明[1,2] GUO ZhiXiong;SUN LengXue;ZHENG JiaMin;CAI CanJun;WANG Bei;LI KaiTuo;PAN TengFei;SHE WenQin;CHEN GuiXin;PAN DongMing(Institute of Postharvest Science and Technology of Horticultural Products,Fujian Agriculture and Forestry University,Fuzhou 350002;College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002)

机构地区：[1]福建农林大学园艺产品贮藏保鲜研究所,福州350002 [2]福建农林大学园艺学院,福州350002

出　　处：《中国农业科学》2021年第16期3502-3513,共12页Scientia Agricultura Sinica

基　　金：福建省科技重大专项(2013NZ0002-1D);福建农林大学科技创新专项基金(KFA17364A)。

摘　　要：【目的】植物第Ⅲ类过氧化物酶具有广泛的生理功能。前期研究发现荔枝果皮具高活性的离子结合态过氧化物酶(BPox),与果实的着色、成熟密切关联,但其特性、作用机制不清楚。明确BPox的生化特性及其基因表达变化,为进一步研究BPox参与荔枝果实着色和成熟机制奠定基础。【方法】以‘乌叶’荔枝成熟果果皮为材料,通过提取及Streamline Phenyl、CM-52、Phenyl Sepharose HP和Superdex 200等柱层析,纯化获得BPox。测定BPox的最适反应pH、最适反应温度、底物特异性、抑制剂等生化特性。采用双倒数法测定其催化愈创木酚、(−)-表儿茶素的Km值及V_(max)。应用MALDI串联质谱鉴定BPox的肽段序列,克隆BPox的cDNA。分别测定盛花后58、69、76、80和90 d荔枝果皮BPox的活性变化,应用荧光定量PCR分析BPox的基因表达变化。【结果】从荔枝果皮纯化得到BPox最主要的2个组分,分别命名为BPox-2和BPox-3。凝胶过滤层析和SDS-PAGE结果显示,BPox-2和BPox-3的表观分子量分别约为30 kD和34 kD。BPox-2和BPox-3的最适反应pH均为6.0,最适反应温度分别为40℃和45℃;二者具有相似的底物特异性;DTT、ASA和L-Cys等能强烈抑制其活性。BPox-2和BPox-3催化愈创木酚的Km值分别为2.97和2.58 mmol·L^(-1),其V_(max)分别为38.72×10^(6)和23.06×10^(6) U·mg^(-1);其催化(−)-表儿茶素的Km值分别为3.49和3.24 mmol·L^(-1),V_(max)分别为38.72×10^(6)和23.06×10^(6) U·mg^(-1)。尽管BPox-2和BPox-3的肽质量指纹(PMF)不同,串联质谱分析显示,二者均具一个序列为TASLSAANSDLPSPFADLATLIAR的胰酶水解肽段。克隆得到BPox2的cDNA,大小为960 bp,共编码319个氨基酸。cDNA编码的多肽链N端包含1段26个氨基酸残基的信号肽,C端缺乏液泡分选序列,具1个潜在的N-糖基化修饰位点。幼果期,荔枝果皮BPox的活性表现弱;至盛花后76 d,果皮开始着色变红,BPox的活性迅速启动升高直至果实成熟。qPCR的�【Objective】In a previous study,the considerable activity of ionically bound peroxidase(BPox)was found in litchi pericarp.The BPox revealed a close relationship with the fruit maturation,but its role was unclear.This work was aimed to elucidate the biochemical properties and gene expression pattern of BPox for the further investigation of its involvement in the process of litchi coloration and maturation.【Method】The mature pericarp of litchi(Litchi chinensis Sonn.cv.Wuye)was used as material,and the BPox was extracted and purified through column chromatography of Streamline Phenyl,CM-52,Phenyl Sepharose and Superdex-200,respectively.The optimal pH and temperature,the substrate specificity and the inhibitors were measured,respectively.The Km values of BPox for guaiacol and(-)-epicatechin and V_(max) values were determined by using double reciprocal plots,respectively.The purified Bpox protein was conducted to SDS-PAGE and in-gel digestion by trypsin,and the sequences of the peptide fragments were identified by using MALDI tandem TOF MS.Total RNA was isolated from litchi pericarp,and the cDNA encoding BPox was cloned.The fruits were harvested 58,69,76,80 and 90 days after full blooming(DAFB),the determination of BPox activity changes in the pericarp and the analysis of BPox gene expression using real-time quantitative PCR,were performed,respectively.【Result】Two most major fractions of ionically bound cationic peroxidase,named BPox-2 and BPox-3,were purified from litchi pericarp,respectively.The apparent molecular weights of the two isoforms were the same,and were estimated to be 30 and 34 kD by gel filtration and SDS-PAGE,respectively.For the BPox-2 and the BPox-3,the optimal pH was 6.0,and the optimal temperature was 40℃and 45℃,respectively.In the presence of H2O2,similar substrate affinity was revealed,while guaiacol and(-)-epicatechin(EC)were the favorable substrates for the two BPoxs.The metal ions test exhibited poor effect on the activity and the most effective inhibitors for litchi BPoxs were

关 键 词：荔枝 结合态过氧化物酶 纯化 质谱鉴定 基因表达 着色和成熟 

分 类 号：S667.1[农业科学—果树学]