Functional Analysis Of Carboxylesterase Gene Family From Populus Trichocarpa

Carboxylesterases(CXE)are a class of hydrolase enzymes with a / ? folding domains,which play key role in plant growth and stress response.The structural and functional investigations of this gene family are of great value for breeding and genetic improvement of forest trees.In this study,we identified the CXE gene family in Populus trichocarpa by TBLASTN search,and then established the gene structure,the distribution of the family memebers on each chromosome,and their expression pattern.We applied affinity chromatography to purify the CXE proteins expressed in E.coli.And then we applied fluorescence assays and p H indicator method to determine the enzymatic activity of CXE proteins.The role of CXE genes in disease resistance was investigated by genetic transformation in Populus tomentosa.This study laid foundation for disease resistance breeding in Populus through genetic manipulation of CXE gene family.The main results are as follows:(1)The CXE gene family in P.trichocarpa was identified,the structural characteristics,genome distribution pattern,gene expression pattern were clarified,and members with tissue-specific expression were identified.A total of 52 CXE genes were identified from the genome of P.trichocarpa,and all 52 CXE proteins contain complete ? / ? hydrolase domains.The results of gene structure analysis showed that 52 CXE genes of P.trichocarpa can be divided into 3 clades,each consists of 39,4 and 9 members,respectively.The chromosome locatizations of these genes show uneven distribution on chromosomes,among which 28 CXE genes are distributed on six chromosomes,and the remaining 24 CXE genes are distributed on 16 chromosomes.The expression analysis indicates that eight CXE genes are not expressed in P.trichocarpa in the five types of tissues examined including xylem,phloem,leaf,twig and root,while the other 44 CXE genes are differentially expressed in these tissues.These results provide a theoretical basis for the study of function and differentiation mechanisms of CXE gene family in P.trichocarpa.(2)The enzymatic activity of the P.trichocarpa CXE proteins was measured in vitro,and the biochemical activities as well as the differences of the five Pt CXE proteins were revealed.Five CXE proteins(Pt CXE4,5,6,41 and 43)were expressed and purified in E.coli.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.Pt CXE5 catalyzed the largest number of natural substrates.The p Hdependent analysis of enzyme activity showed that the catalytic activity of the five proteins increased with the increase of p H in the range of p H 6.0?9.0.This study provides new insights into the functions of carboxylesterase gene family.It provides an important reference for predicting the catalytic ability of CXE genes in plants.(3)The preliminary screening and verification of CXE gene involved in disease resistance were carried out in P.tomentosa.The genetic transformation of six CXE genes of the variety 741 of P.tomentosa produced at least four positive lines for each gene.This promising result will guide future genetic efforts for disease resistance breeding through transgenic positive plants.This study systematically established the gene structure,genome distribution and function of CXE gene family in P.trichocarpa by combining sequence analyses,expression pattern,protein biochemical activity and in vivo function.The results are of valuable for understanding the function of CXE gene family in plants and breeding for disease resistant trees.