Transient Gene Expression Research Articles

Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells.

Membrane proteins are difficult biomolecules to express and purify. In this paper, we compare the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The target proteins were C terminally fused to the green fluorescent marker protein GFP to enable sensitive monitoring. We show that the choice of expression systems makes a considerable difference to the yield and quality of the six selected membrane proteins. Virus-free transient gene expression (TGE) in insect High Five cells combined with solubilization in dodecylmaltoside plus cholesteryl hemisuccinate generated the most homogeneous samples for all six targets. Further, the affinity purification of the solubilized proteins using the Twin-Strep® tag improved protein quality in terms of yield and homogeneity compared to His-tag purification. TGE in High Five insect cells offers a fast and economically attractive alternative to the established methods that require either baculovirus construction and the infection of the insect cells or relatively expensive transient gene expression in mammalian cells for the production of integral membrane proteins.

Application of a novel strong promoter from Chinese fir (Cunninghamia lanceolate) in the CRISPR/Cas mediated genome editing of its protoplasts and transgenesis of rice and poplar.

Novel constitutive promoters are essential for plant biotechnology. Although in angiosperms, a number of promoters were applied in monocots or dicots genetic engineering, only a few promoters were used in gymnosperm. Here we identified two strong promoters (Cula11 and Cula08) from Chinese fir (C. lanceolate) by screening the transcriptomic data and preliminary promoter activity assays in tobacco. By using the newly established Chinese fir protoplast transient expression technology that enables in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII promoters were introduced into Chinese fir protoplasts, and CRISPR/Cas mediated gene knock-out event was successfully achieved. More importantly, compared with the commonly used promoters in the genetic engineering in angiosperms, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi promoter in transgenic rice, respectively, indicating its potential application in poplar and rice genetic engineering. Overall, the novel putative constitutive gene promoters reported here will have great potential application in gymnosperm and angiosperm biotechnology, and the transient gene expression system established here will serve as a useful tool for the molecular and genetic analyses of Chinese fir genes.

Efficiency of Promoters of Human Genes FAP and CTGF at Organism Level in a Danio rerio Model.

The identification of tissue-specific promoters for gene therapeutic constructs is one of the aims of complex tumor therapy. The genes encoding the fibroblast activation protein (FAP) and the connective tissue growth factor (CTGF) can function in tumor-associated stromal cells but are practically inactive in normal adult cells. Accordingly, the promoters of these genes can be used to develop vectors targeted to the tumor microenvironment. However, the efficiency of these promoters within genetic constructs remains underexplored, particularly, at the organism level. Here, we used the model of Danio rerio embryos to study the efficiency of transient expression of marker genes under the control of promoters of the FAP, CTGF, and immediate early genes of Human cytomegalovirus (CMV). Within 96 h after the injection of vectors, the CTGF and CMV promoters provided similar equal efficiency of reporter protein accumulation. In the case of the FAP promoter, a high level of reporter protein accumulation was observed only in certain zebrafish individuals that were considered developmentally abnormal. Disturbed embryogenesis was the factor of changes in the exogenous FAP promoter function. The data obtained make a significant contribution to understanding the function of the human CTGF and FAP promoters within vectors to assess their potential in gene therapy.

Particle bombardment-assisted peptide-mediated gene transfer for highly efficient transient assay

ObjectiveA centrifugation-assisted peptide-mediated gene transfer (CAPT) method was recently developed as an efficient system for gene delivery into plant cells. However, the gene transfer efficiency of CAPT into plant cells was not entirely satisfactory for detecting transient expression of a transgene driven into mitochondria. Here, we report a new gene delivery system using a method called particle bombardment-assisted peptide-mediated gene transfer (PBPT).ResultsWe investigated various parameters of the PBPT method to increase transient gene expression efficiency in Brassica campestris. The optimal conditions for PBPT were a single bombardment with gold particles coated with a DNA‒peptide complex (6 µg of DNA and 2 µg of peptide) at an acceleration pressure of 5 kg/cm2 and a target distance of 12.5 cm. Moreover, bombardment under the optimal conditions successfully transferred the transgene into the cells of other plant species, namely B. juncea and tomato. Thus, we developed a PBPT method for highly efficient delivery of a DNA‒peptide complex into plant mitochondria.

Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.

Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.

Depletion of the NbCORE receptor drastically improves agroinfiltration productivity in older Nicotiana benthamiana plants.

Nicotiana benthamiana is increasingly used for transient gene expression to produce antibodies, vaccines, and other pharmaceutical proteins but transient gene expression is low in fully developed, 6-8-week old plants. This low gene expression is thought to be caused by the perception of the cold shock protein (CSP) of Agrobacterium tumefaciens. The CSP receptor is contested because both NbCSPR and NbCORE have been claimed to perceive CSP. Here, we demonstrate that CSP perception is abolished in 6-week-old plants silenced for NbCORE but not NbCSPR. Importantly, older NbCORE-silenced plants support a highly increased level of GFP fluorescence and protein upon agroinfiltration. The drastic increase in transient protein production in NbCORE-depleted plants offers new opportunities for molecular farming, where older plants with larger biomass can now be used for efficient protein expression.

Practical use of tobravirus-based vector to produce SARS-CoV-2 antigens in plants

A plant-based heterologous expression system is an attractive option for recombinant protein production because it is based on a eukaryotic system of high feasibility, and low biological risks. Frequently, binary vector systems are used for transient gene-expression in plants. However, plant virus vector-based systems offer advantages for higher protein yields due to their self-replicating machinery. In the present study, we show an efficient protocol using a plant virus vector based on a tobravirus, pepper ringspot virus, that was employed for transient expression of severe acute respiratory syndrome coronavirus 2 partial gene fragments of the spike (named S1-N) and the nucleocapsid (named N) proteins in Nicotiana benthamiana plants. Purified proteins yield of 40–60 µg/g of fresh leaves were obtained. Both proteins, S1-N and N, showed high and specific reactivities against convalescent patients’ sera by the enzyme-linked immunosorbent assay format. The advantages and critical points in using this plant virus vector are discussed.

Development of an efficient method for selection of stable cell pools for protein expression and surface display with Expi293F cells.

Compare with transient expression, stable cell lines generally have higher productivity and better quality for protein expression. However, selection of stable cell line is time-consuming and laborious. Here we describe an optimized selection method to achieve high-efficient stable cell pools with Expi293F suspension cells. This method only takes 2-3 weeks to generate stable cell pools with 2- to 10-fold higher productivity than transient gene expression (TGE). In fed-batch culture with Yeastolate, >1 g/L yield was achieved with our KTN0239-IgG stable cell pool in shaker flasks. This method can be also applied to efficiently display proteins on the cell surface.

The Role of MbEGS1 and MbEGS2 in Methyleugenol Biosynthesis by Melaleuca bracteata

Many aromatic plant volatile compounds contain methyleugenol, which is an attractant for insect pollination and has antibacterial, antioxidant, and other properties. The essential oil of Melaleuca bracteata leaves contains 90.46% methyleugenol, which is an ideal material for studying the biosynthetic pathway of methyleugenol. Eugenol synthase (EGS) is one of the key enzymes involved in the synthesis of methyleugenol. We recently reported two eugenol synthase genes (MbEGS1 and MbEGS2) present in M. bracteata, where MbEGS1 and MbEGS2 were mainly expressed in flowers, followed by leaves, and had the lowest expression levels in stems. In this study, the functions of MbEGS1 and MbEGS2 in the biosynthesis of methyleugenol were investigated using transient gene expression technology and virus-induced gene silencing (VIGS) technology in M. bracteata. Here, in the MbEGSs genes overexpression group, the transcription levels of the MbEGS1 gene and MbEGS2 gene were increased 13.46 times and 12.47 times, respectively, while the methyleugenol levels increased 18.68% and 16.48%. We further verified the function of the MbEGSs genes by using VIGS, as the transcript levels of the MbEGS1 and MbEGS2 genes were downregulated by 79.48% and 90.35%, respectively, and the methyleugenol content in M. bracteata decreased by 28.04% and 19.45%, respectively. The results indicated that the MbEGS1 and MbEGS2 genes were involved in the biosynthesis of methyleugenol, and the transcript levels of the MbEGS1 and MbEGS2 genes correlated with the methyleugenol content in M. bracteata.

Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis

Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.

Dual-omics reveals temporal differences in acute sympathetic stress-induced cardiac inflammation following α1 and β-adrenergic receptors activation.

Sympathetic stress is prevalent in cardiovascular diseases. Sympathetic overactivation under strong acute stresses triggers acute cardiovascular events including myocardial infarction (MI), sudden cardiac death, and stress cardiomyopathy. α1-ARs and β-ARs, two dominant subtypes of adrenergic receptors in the heart, play a significant role in the physiological and pathologic regulation of these processes. However, little is known about the functional similarities and differences between α1- and β-ARs activated temporal responses in stress-induced cardiac pathology. In this work, we systematically compared the cardiac temporal genome-wide profiles of acute α1-AR and β-AR activation in the mice model by integrating transcriptome and proteome. We found that α1- and β-AR activations induced sustained and transient inflammatory gene expression, respectively. Particularly, the overactivation of α1-AR but not β-AR led to neutrophil infiltration at one day, which was closely associated with the up-regulation of chemokines, activation of NF-κB pathway, and sustained inflammatory response. Furthermore, there are more metabolic disorders under α1-AR overactivation compared with β-AR overactivation. These findings provide a new therapeutic strategy that, besides using β-blocker as soon as possible, blocking α1-AR within one day should also be considered in the treatment of acute stress-associated cardiovascular diseases.

Development of a Molasses-Based Medium for Agrobacterium tumefaciens Fermentation for Application in Plant-Based Recombinant Protein Production

The Agrobacterium-mediated transient gene expression system is a rapid and efficient method for heterologous recombinant protein expression in plants. The fermentation of genetically modified Agrobacterium tumefaciens is an important step in increasing the efficiency of recombinant protein production in plants. However, the limitation of this system that makes it economically non-competitive for industrial-scale applications is the Agrobacterium suspension production cost. In this study, the utilization of sugarcane molasses as an alternative low-cost source of carbon at a concentration of 8.7 g/L and nitrogen at a concentration of 2.4 g/L for Agrobacterium cultivation was investigated. Molasses pretreatment using sulfuric acid (SA) was applied before fermentation, and it resulted in a maximum specific growth rate of 0.232 ± 0.0063 h−1 in the A. tumefaciens EHA105 culture. The supplementation of antibiotics in the molasses-based medium was shown to be unnecessary for plasmid maintenance during fermentation in both Agrobacterium strains, which helped to reduce the production cost. We evaluated recombinant protein production using an Agrobacterium culture without antibiotic supplementation in the growth media by demonstrating green fluorescent protein expression in wild-type Nicotiana benthamiana leaves. In the evaluation of the culture medium cost, the molasses-based medium cost was 6.1 times lower than that of LB. Finally, this study demonstrated that the newly developed molasses-based medium for Agrobacterium fermentation is a feasible and effective medium for transient recombinant protein production in plant tissues.

Development of stable HEK293T cell pools expressing CSFV E2 protein: A potential antigen expression platform.

Large quantities of antigens are required since protective antigens, such as classical swine fever virus (CSFV) E2 protein, are widely used in diagnostic reagents and subunit vaccines. Compared to clonal cell lines and transient gene expression, stable cell pools provide a potential alternative platform to rapidly produce large amounts of antigens. In this work, firstly, Human embryonic kidney 293T (HEK293T) cell pools expressing E2 protein were developed by transduction of lentiviral vectors. On the one hand, the SP7 was selected from 7 well-performing signal peptides to remarkably increase the production of E2 protein. On the other hand, it was found that high MOI could improve the expression of E2 protein by increasing gene copy numbers. Moreover, the HEK293T cell pools were evaluated for stability by passages and batch cultures, demonstrating that the cell pools were stable for at least 90days. And then, the performance of the cell pools in batch, fed-batch, and semi-perfusion was studied. Among them, the titer of E2 protein was up to 2g/L in semi-perfusion, which is currently the highest to the authors' knowledge. Finally, the aggregations and immunogenicity of the E2 protein were analyzed by SDS-PAGE and immunization of mice, respectively. There was no significant difference in aggregations and antibody titers of E2 protein in three culture methods. These results suggest that stable HEK293T cell pools are a promising and robust platform for rapid and efficient production of recombinant proteins.

The Basic Experimental Setup for the Vacuum-assisted, Agrobacterium-mediated Transient Expression in Tomato

Several methods are available for use, to deliver the gene of interest into plants. Among these, Agrobacterium-mediated transformation utilizes binary vector systems to achieve the stable transformation of plants. Alas, this process is labor-intensive and time-consuming as several months are needed to obtain a true transgenic plant. Transient gene expression (e.g., vacuum-infiltration) systems were offered as an alternative over stable transformation, specifically to overcome time-related drawbacks. However, this method requires expensive equipment such as vacuum chambers. In this study, we report a vacuum-infiltration protocol for the transient expression of a reporter gene, modified green fluorescent protein (mGFP), in tomato seedling. With a basic experimental setup (including a custom-built growth chamber) with affordable equipment, we showed that the entire leaf can be infiltrated by applying a mere 200 millibar vacuum for 6 minutes, and thus the transient expression can be achieved in tomato plant, evidenced by PCR-based detection of the T-DNA, detection of mGFP both analytically with SDS-page analysis, and visually by the images acquired by fluorescence microscopy. Furthermore, different Agrobacterium tumefaciens strains were tested for their transformation efficiency, and we showed that LBA4404 was the most effective strain to use in the vacuum-assisted transient expression.

Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis

BackgroundCotton has tremendous economic value worldwide; however, its allopolyploid nature and time-consuming transformation methods have hampered the development of cotton functional genomics. The protoplast system has proven to be an important and versatile tool for functional genomics, tissue-specific marker gene identification, tracking developmental trajectories, and genome editing in plants. Nevertheless, the isolation of abundant viable protoplasts suitable for single-cell RNA sequencing (scRNA-seq) and genome editing remains a challenge in cotton.ResultsWe established an efficient transient gene expression system using protoplasts isolated from cotton taproots. The system enables the isolation of large numbers of viable protoplasts and uses an optimized PEG-mediated transfection protocol. The highest yield (3.55 × 105/g) and viability (93.3%) of protoplasts were obtained from cotton roots grown in hydroponics for 72 h. The protoplasts isolated were suitable for scRNA-seq. The highest transfection efficiency (80%) was achieved when protoplasts were isolated as described above and transfected with 20 μg of plasmid for 20 min in a solution containing 200 mM Ca2+. Our protoplast-based transient expression system is suitable for various applications, including validation the efficiency of CRISPR vectors, protein subcellular localization analysis, and protein–protein interaction studies.ConclusionsThe protoplast isolation and transfection protocol developed in this study is stable, versatile, and time-saving. It will accelerate functional genomics and molecular breeding in cotton.

Carotenoid metabolism: New insights and synthetic approaches.

Carotenoids are well-known isoprenoid pigments naturally produced by plants, algae, photosynthetic bacteria as well as by several heterotrophic microorganisms. In plants, they are synthesized in plastids where they play essential roles in light-harvesting and in protecting the photosynthetic apparatus from reactive oxygen species (ROS). Carotenoids are also precursors of bioactive metabolites called apocarotenoids, including vitamin A and the phytohormones abscisic acid (ABA) and strigolactones (SLs). Genetic engineering of carotenogenesis made possible the enhancement of the nutritional value of many crops. New metabolic engineering approaches have recently been developed to modulate carotenoid content, including the employment of CRISPR technologies for single-base editing and the integration of exogenous genes into specific "safe harbors" in the genome. In addition, recent studies revealed the option of synthetic conversion of leaf chloroplasts into chromoplasts, thus increasing carotenoid storage capacity and boosting the nutritional value of green plant tissues. Moreover, transient gene expression through viral vectors allowed the accumulation of carotenoids outside the plastid. Furthermore, the utilization of engineered microorganisms allowed efficient mass production of carotenoids, making it convenient for industrial practices. Interestingly, manipulation of carotenoid biosynthesis can also influence plant architecture, and positively impact growth and yield, making it an important target for crop improvements beyond biofortification. Here, we briefly describe carotenoid biosynthesis and highlight the latest advances and discoveries related to synthetic carotenoid metabolism in plants and microorganisms.

In-planta transient transformation of avocado (Persea americana) by vacuum agroinfiltration of aerial plant parts

Avocado, Persea americana Mill, is one of the most traded tropical fruits in the international market. To date, stable and transient transformation has only been achieved for of zygotic embryos and not of adult plant tissue, which limits functional genomics research. We provide the first transient Agrobacterium-mediated transformation methodology in avocado leaves that overcomes the recalcitrance to transformation of this species. We investigated the effect of Agrobacterium strain, leaf stage, wounding pre-treatment, the phytohormone jasmonic acid, and vacuum infiltration on transient transformation of avocado leaves. Using the Agrobacterium strain LBA4404 and the RUBY reporter a transformation frequency of up to 27% was obtained for avocado detached leaves. The transformation efficiency depended on the age of the leaf, with an intermediate stage of leaf development showing the highest efficiency of transient reporter gene expression. Microwounding pre-treatment facilitates agroinfiltration and coupled with leaf age are the primary factors influencing competence for transient transformation. Jasmonic acid did not significantly affect transient transformation in the absence of microwounding. However, microwounding and 250 µM of jasmonic acid acted synergistically to significantly enhance transient expression. Using this methodology with localized vacuum agroinfiltration, transient transformation of attached avocado leaves was achieved. This method unlocks the use of Agrobacterium-mediated transient transformation as a tool for explore gene function and metabolic pathways in both, detached and attached avocado leaves.

Inoculation of Maize with Sugarcane Mosaic Virus Constructs and Application for RNA Interference in Fall Armyworms.

Virus-mediated transient gene overexpression and gene expression silencing can be used to screen gene functions in plants. Sugarcane mosaic virus (SCMV) is a positive strand RNA virus in the Potyviridae family that has been modified to be used as vector to infect monocots, including maize (Zea mays), for transient gene overexpression and gene expression silencing. Relative to stable transformation, SCMV-mediated transient expression in maize has the advantages of being faster and less expensive. Here, we describe a protocol for cloning constructs into the plasmid vector pSCMV-CS3. After maize seedlings are transformed with pSCMV-CS3 constructs by particle bombardment, the virus replicates and spreads systemically in the plants. Subsequent infections of maize seedlings can be accomplished by rub inoculation with sap from SCMV-infested plants. As an example of a practical application of the method, we also describe virus-induced gene silencing (VIGS) of fall armyworm (Spodoptera frugiperda) gene expression. Transgenic viruses are created by cloning a segment of the fall armyworm target gene into pSCMV-CS3 prior to maize transformation. Caterpillars are fed on the virus-infected maize plants, which make dsRNA to silence the expression of the fall armyworm target gene after ingestion. This use of SCMV for plant-mediated VIGS in insects allows rapid screening of gene functions when caterpillars are feeding on their host plants. Graphical overview.

Efficient Transient Expression for Functional Analysis in Fruit Using the Tsukuba System Vector

Evaluating the function of genes expressed in fruit tissues of fruit tree species using a genetic transformation approach is a long process because the trees are generally recalcitrant to genetic transformation and cannot bear fruit during their long juvenile phases. Transient gene expression in fruit enables the functional analysis of genes associated with fruit traits, which may accelerate the study of fruit physiology. Here, by using the recently developed “Tsukuba system”, we successfully established an efficient transient expression system in harvested fruit tissues. The “Tsukuba system” utilizes a combination of the geminiviral replication system and a double terminator, which ensures sufficient levels of transgene expression. We used blueberry fruit as a model to characterize the applicability of this system for transient expression in fruit tissue. The pTKB3-EGFP vector was introduced by agroinfiltration into the fruit tissues of several blueberry cultivars. We found that transient GFP fluorescence in fruit peaked 4–6 days after agroinfiltration. Agrobacterium suspensions were easily injected into soft, mature fruit, and GFP was strongly expressed; however, hard, immature fruit were not penetrable by Agrobacterium suspensions, and GFP was rarely detected. We then tested the applicability of the developed system to other fruit tree species: six families, 17 species, and 26 cultivars. GFP fluorescence was detected in all species, except for Japanese apricot. In blueberry, bilberry, sweet cherry, apricot, and satsuma mandarin, GFP was highly expressed and observed in a large proportion of the flesh. In kiwifruit, hardy kiwifruits, persimmon, peach, apple, European pear, and grape, GFP fluorescence was limited to certain parts of the fruits. Finally, transient VcMYBA1 overexpression in blueberry was tested as a model for gene functional analysis in fruit. Transient VcMYBA1 overexpression induced red pigmentation in the flesh, suggesting that VcMYBA1 expression caused anthocyanin accumulation. This study provides a technical basis for the rapid evaluation of genes expressed in fruit, which will be useful for gene function evaluation studies in fruit crops with long juvenile phases.

The Functional Characterization of Carboxylesterases Involved in the Degradation of Volatile Esters Produced in Strawberry Fruits.

Volatile ester compounds are important contributors to the flavor of strawberry, which affect consumer preference. Here, the GC-MS results showed that volatile esters are the basic aroma components of strawberry, banana, apple, pear, and peach, and the volatile esters were significantly accumulated with the maturation of strawberry fruits. The main purpose of this study is to discuss the relationship between carboxylesterases (CXEs) and the accumulation of volatile ester components in strawberries. FaCXE2 and FaCXE3 were found to have the activity of hydrolyzing hexyl acetate, Z-3-hexenyl acetate, and E-2-hexenyl acetate to the corresponding alcohols. The enzyme kinetics results showed that FaCXE3 had the higher affinity for hexyl acetate, E-2-hexenyl acetate, and Z-3-hexenyl acetate compared with FaCXE2. The volatile esters were mainly accumulated at the maturity stages in strawberry fruits, less at the early stages, and the least during the following maturation stages. The expression of FaCXE2 gradually increased with fruit ripening and the expression level of FaCXE3 showed a decreasing trend, which suggested the complexity of the true function of CXEs. The transient expression of FaCXE2 and FaCXE3 genes in strawberry fruits resulted in a significantly decreased content of volatile esters, such as Z-3-hexenyl acetate, methyl hexanoate, methyl butyrate, and other volatile esters. Taken together, FaCXE2 and FaCXE3 are indeed involved in the regulation of the synthesis and degradation of strawberry volatile esters.