Abstract:Carboxylesterases (CXE) are a class of hydrolase enzymes with α/β folding 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 complete α/β hydrolase 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 4MU acetate and 4MU butyrate, but none of them can catalyze the hydrolysis of 4MU laurate and 4MU palmitate. The CXE showed lower catalytic activity towards substrates with longer carbon chains. The pHdependent 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.