毛果杨羧酸酯酶基因家族的功能研究

Functional Analysis of Carboxylesterase Gene Family from Populus trichocarpa

羧酸酯酶(CXE)是一类具有α/β折叠结构域的水解酶类,在植物生长发育和胁迫响应中发挥着重要作用。该研究以毛果杨(Populus trichocarpa)植株为材料,通过TBLASTN搜索和手动校正鉴定毛果杨CXE基因,并分析CXE基因家族基本特征、基因结构和表达模式,通过亲和层析纯化大肠杆菌表达的毛果杨CXE蛋白,然后利用荧光法和pH指示剂法测定CXE蛋白酶学活性,为深入揭示林木CXE基因家族的功能奠定基础。结果表明:(1)从毛果杨基因组中共鉴定出52个CXE基因,且52个CXE蛋白都含有完整的α/β水解酶结构域;毛果杨52个CXE基因分为3个类型,每一类型分别有39、4和9个CXE基因。(2)毛果杨CXE基因在染色体上的分布不均匀,其中有28个CXE基因以基因簇的形式分布在6条染色体上,其余24个CXE基因分散分布在16个染色体上。(3)毛果杨8个CXE基因在木质部、韧皮部、叶、小枝和根等5个组织部位均未检测到表达,其余44个CXE基因在5个组织部位呈现差异性表达。(4)在大肠杆菌中表达并纯化了5个毛果杨CXE蛋白(PtCXE4、5、6、41、43),且5个蛋白对植物体内发现的24个天然底物的催化能力测定发现,PtCXE5可以催化天然底物的个数最多(11个);酶学性质分析表明,5个蛋白都可以催化4-甲基伞形酮乙酸酯(4-MU acetate)和4-甲基伞形酮丁酸酯(4-MU butyrate)水解,却都不能催化4-甲基伞形酮月桂酸酯(4-MU laurate)和4-甲基伞形酮棕榈酸酯(4-MU palmitate)水解,且随着底物碳链长度的增加,蛋白的催化活性也随之下降;酶活性的pH依赖性分析发现,在pH6.0~9.0范围内,随着pH的升高5个蛋白的催化活性均呈现升高的趋势。

Carboxylesterases(CXE)are a class of hydrolase enzymes withα/βfolding domains,which play key roles in plant growth and stress response.In this study,Populus trichocarpa was used as material.The CXE genes of P.trichocarpa were identified by TBLASTN search and manual correction,and then the basic characteristics,gene structure and expression pattern of the CXE gene family were analyzed.The P.trichocarpa CXE proteins expressed in E.coli were purified by affinity chromatography,and then the CXE activity was determined by fluorescence method and pH indicator method,respectively.This study laid foundation for further revealing the function of the forest tree CXE gene family.The results showed that:(1)a total of 52 CXE genes were identified from the P.trichocarpa genome,and the 52 CXE proteins all contained a completeα/βhydrolase domain.The 52 P.trichocarpa CXE genes were divided into 3 types,each had 39,4 and 9 CXE genes,respectively.(2)The distribution of CXE genes on the chromosomes of P.trichocarpa is uneven.Among them,28 CXE genes are distributed on 6 chromosomes in the form of gene clusters,and the remaining 24 CXE genes are scattered on 16 chromosomes.(3)Among all the 52 CXEs,expression of eight CXE genes were not detectable in the xylem,phloem,leaf,shooting and root of P.trichocarpa,while the other 44 CXE genes were differentially expressed in the five tissues.(4)Five CXE proteins(PtCXE4,5,6,41,and 43)were expressed in E.coli,and then purified to detect activity.PtCXE5 was found to catalyze the largest number of natural substrates.All the five purified proteins can catalyze the hydrolysis of 4-MU acetate and 4-MU butyrate,but none of them can catalyze the hydrolysis of 4-MU laurate and 4-MU palmitate.The CXE showed lower catalytic activity towards substrates with longer carbon chains.The pH-dependent analysis of enzyme activity showed that the catalytic activities of the five proteins increased with the increase of pH in the range of pH 6.0-9.0.This study provides new insights into the functions of carboxylesterase gene family.