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Background: In plant genomes, high proportions of duplicate copies reveals that gene duplications play an important role in the evolutionary processes of plant species. A series of gene families under positive selection after recent duplication events in plant genomes indicated the evolution of duplicates driven by adaptive evolution. However, the genome-wide evolutionary features of young duplicate genes among closely related species are rarely reported. Results: In this study, we conducted a systematic survey of young duplicate genes at genome-wide levels among six Rosaceae species, whose whole-genome sequencing data were successively released in recent years. A total of 35,936 gene families were detected among the six species, in which 60.25% were generated by young duplications. The 21,650 young duplicate gene families could be divided into two expansion types based on their duplication patterns, species-specific and lineage-specific expansions. Our results showed the species-specific expansions advantaging over the lineage-specific expansions. In the two types of expansions, high-frequency duplicate domains exhibited functional preference in response to environmental stresses. Conclusions: The functional preference of the young duplicate genes in both the expansion types showed that they were inclined to respond to abiotic or biotic stimuli. Moreover, young duplicate genes under positive selection in both species-specific and lineage-specific expansions suggested that they were generated to adapt to the environmental factors in Rosaceae species. [ABSTRACT FROM AUTHOR]

Transcription factors regulate gene expression in response to various external and internal cues by activating or suppressing downstream genes. Significant progress has been made in identifying and characterizing the Cysteine3Histidine (C3H) gene family in several dicots and monocots. They are characterized by their signature motif of three cysteine and one histidine residues, and reportedly play important roles in regulation of plant growth, developmental processes and environmental responses. In this study, we performed genome-wide and deep analysis of putative C3H genes, and a total of 117 PbeC3H members, were identified in P. betulaefolia and classified into 12 groups. Results were supported by the gene structural characteristics and phylogenetic analysis. These genes were unevenly distributed on 17 chromosomes. The gene structures of the C3H genes were relatively complex but conserved in each group. The C3H genes experienced a WGD event that occurred in the ancestor genome of P. betulaefolia and apple before their divergence based on the synonymous substitutions (Ks) values. There were 35 and 37 pairs of paralogous genes in the P. betulaefolia and apple genome, respectively, and 87 pairs of orthologous genes between P. betulaefolia and apple were identified. Except for one orthologous pairs PbeC3H66 and MD05G1311700 which had undergone positive selection, the other C3H genes had undergone purifying selection. Expression profiles showed that high salinity stress could influence the expression level of C3H genes in P. betulaefolia. Four members were responsive to salt stress in roots, nine were responsive to salt stress in leaves and eight showed inhibited expression in leaves. Results suggested important roles of PbeC3H genes in response to salt stress and will be useful for better understanding the complex functions of the C3H genes, and will provide excellent candidates for salt-tolerance improvement. [ABSTRACT FROM AUTHOR]

Dehydration-responsive element binding proteins are transcription factors that play a critical role in plant response to temperature stress. Over-expression of DREB genes has been demonstrated to enhance temperature stress tolerance. A series of physiological and biochemical modifications occur in a complex and integrated way when plants respond to temperature stress, which makes it difficult to assess the mechanism underlying the DREB enhancement of stress tolerance. A meta-analysis was conducted of the effect of DREB overexpression on temperature stress tolerance and the various parameters modulated by overexpression that were statistically quantified in 75 published articles. The meta-analysis was conducted to identify the overall influence of DREB on stress-related parameters in transgenic plants, and to determine how different experimental variables affect the impact of DREB overexpression. Viewed across all the examined studies, 7 of the 8 measured plant parameters were significantly (p = 0.05) modulated in DREB-transgenic plants when they were subjected to temperature stress, while 2 of the 8 parameters were significantly affected in non-stressed control plants. The measured parameters were modulated by 32% or more by various experimental variables. The modulating variables included, acclimated or non-acclimated, type of promoter, stress time and severity, source of the donor gene, and whether the donor and recipient were the same genus. These variables all had a significant effect on the observed impact of DREB overexpression. Further studies should be conducted under field conditions to better understand the role of DREB transcription factors in enhancing plant tolerance to temperature stress. [ABSTRACT FROM AUTHOR]

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Background: Plant disease resistance (R) genes are evolving rapidly and play a critical role in the innate immune system of plants. The nucleotide binding sites-leucine rich repeat (NBS-LRR) genes are one of the largest classes in plant R genes. Previous studies have focused on the NBS-LRR genes from one or several species of different genera, and the sequenced genomes of the genus Fragaria offer the opportunity to study the evolutionary processes of these R genes among the closely related species. Results: In this study, 325, 155, 190, 187, and 133 NBS-LRRs were discovered from F. x ananassa, F. iinumae, F. nipponica, F. nubicola, and F. orientalis, respectively. Together with the 144 NBS-LRR genes from F. vesca, a total of 1134 NBS-LRRs containing 866 multi-genes comprised 184 gene families across the six Fragaria genomes. Extremely short branch lengths and shallow nodes were widely present in the phylogenetic tree constructed with all of the NBS-LRR genes of the six strawberry species. The identities of the orthologous genes were highly significantly greater than those of the paralogous genes, while the Ks ratios of the former were very significantly lower than those of the latter in all of the NBS-LRR gene families. In addition, the Ks and Ka/Ks values of the TIR-NBS-LRR genes (TNLs) were significantly greater than those of the non-TIR-NBS-LRR genes (non-TNLs). Furthermore, the expression patterns of the NBS-LRR genes revealed that the same gene expressed differently under different genetic backgrounds in response to pathogens. Conclusions: These results, combined with the shared hotspot regions of the duplicated NBS-LRRs on the chromosomes, indicated that the lineage-specific duplication of the NBS-LRR genes occurred before the divergence of the six Fragaria species. The Ks and Ka/Ks ratios suggested that the TNLs are more rapidly evolving and driven by stronger diversifying selective pressures than the non-TNLs. [ABSTRACT FROM AUTHOR]

Asian pear (Pyrus bretschneideri) is one of the most important fruit crops in the world, and its growth and productivity are frequently affected by abiotic stresses. Calcineurin B-like interacting protein kinases (CIPKs) as caladium-sensor protein kinases interact with Ca2+-binding CBLs to extensively mediate abiotic stress responses in plants. Although the pear genome sequence has been released, little information is available about the CIPK genes in pear, especially in response to salt and osmotic stresses. In this study, we systematically identified 28 CIPK family members from the sequenced pear genome and analyzed their organization, phylogeny, gene structure, protein motif, and synteny duplication divergences. Most duplicated PbCIPKs underwent purifying selection, and their evolutionary divergences accompanied with the pear whole genome duplication. We also investigated stress -responsive expression patterns and co-expression networks of CIPK family under salt and osmotic stresses, and the distribution of stress-related cis-regulatory elements in promoter regions. Our results suggest that most PbCIPKs could play important roles in the abiotic stress responses. Some PbCIPKs, such as PbCIPK22, -19, -18, -15, -8, and -6 can serve as core regulators in response to salt and osmotic stresses based on co-expression networks of PbCIPKs. Some sets of genes that were involved in response to salt did not overlap with those in response to osmotic responses, suggesting the sub-functionalization of CIPK genes in stress responses. This study revealed some candidate genes that play roles in early responses to salt and osmotic stress for further characterization of abiotic stress responses medicated by CIPKs in pear. [ABSTRACT FROM AUTHOR]

Background: Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs or multiple transformation events that is often restricted by a lack of available transformation markers. Results: Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven nonfamily genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. Conclusions: We present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering. [ABSTRACT FROM AUTHOR]

Background Mitogen-activated protein kinase kinase kinases (MAPKKKs; MAP3Ks) are important components of MAPK cascades, which are highly conserved signal transduction pathways in animals, yeast and plants, play important roles in plant growth and development. MAPKKKs have been investigated on their evolution and expression patterns in limited plants including Arabidopsis, rice and maize.In the phylogenetic analysis, MAPKKK genes of grapevine have been classified into three subgroups as described for Arabidopsis, named MEKK, ZIK and RAF, also confirmed in grapevine by the analysis of conserved motifs and exon-intron organizations. By analyzing expression profiles of MAPKKK genes in grapevine microarray databases, we highlighted the modulation of different MAPKKKs in different organs and distinct developmental stages. Furthermore, we experimentally investigated the expression profiles of 45 grape MAPKKK genes in response to biotic (powdery mildew) and abiotic stress (drought), as well as to hormone (salicylic acid, ethylene) and hydrogen peroxide treatments, and identified several candidate MAPKKK genes that might play an important role in biotic and abiotic responses in grapevine, for further functional characterization. Conclusions This is the first comprehensive experimental survey of the grapevine MAPKKK gene family, which provides insights into their potential roles in regulating responses to biotic and abiotic stresses, and the evolutionary expansion of MAPKKKs is associated with the diverse requirement in transducing external and internal signals into intracellular actions in MAPK cascade in grapevine. [ABSTRACT FROM AUTHOR]

Background: The traditional phylogeny analysis within gene family is mainly based on DNA or amino acid sequence homologies. However, these phylogenetic tree analyses are not suitable for those "non-traditional" gene families like microRNA with very short sequences. For the normal protein-coding gene families, low bootstrap values are frequently encountered in some nodes, suggesting low confidence or likely inappropriateness of placement of those members in those nodes. Results: We introduce MicroSyn software as a means of detecting microsynteny in adjacent genomic regions surrounding genes in gene families. MicroSyn searches for conserved, flanking colinear homologous gene pairs between two genomic fragments to determine the relationship between two members in a gene family. The colinearity of homologous pairs is controlled by a statistical distance function. As a result, gene duplication history can be inferred from the output independent of gene sequences. MicroSyn was designed for both experienced and non-expert users with a user-friendly graphical-user interface. MicroSyn is available from: http://fcsb.njau.edu. cn/microsyn/. Conclusions: Case studies of the microRNA167 genes in plants and Xyloglucan ndotransglycosylase/Hydrolase family in Populus trichocarpa were presented to show the utility of the software. The easy using of MicroSyn in these examples suggests that the software is an additional valuable means to address the problem intrinsic in the computational methods and sequence qualities themselves in gene family analysis. [ABSTRACT FROM AUTHOR]

Antibiotic-resistance genes of bacterial origin are invaluable markers for plant genetic engineering. However, these genes are feared to pose possible risk to human health by horizontal gene transfer from transgenic plants to bacteria, potentially resulting in antibiotic-resistant pathogenic bacteria; this is a considerable regulatory concern in some countries. The Atwbc19 gene, encoding an Arabidopsis thaliana ATP-binding cassette transporter, has been reported to confer resistance to kanamycin specifically as an alternative to bacterial antibiotic-resistance genes. In this report, we transformed hybrid aspen ( Populus canescens × P. grandidentata) with the Atwbc19 gene. Unlike Atwbc19-transgenic tobacco that was only resistant to kanamycin, the transgenic Populus plants also showed resistance to three other aminoglycoside antibiotics (neomycin, geneticin, and paromomycin) at comparable levels to plants containing a CaMV35S- nptII cassette. Although it is unknown why the transgenic Populus with the Atwbc19 gene is resistant to all aminoglycoside antibiotics tested, the broad utility of the Atwbc19 gene as a reporter gene is confirmed here in a second dicot species. Because the Atwbc19 gene is plant-ubiquitous, it might serve as an alternative selectable marker to current bacterial antibiotic-resistance marker genes and alleviate the potential risk for horizontal transfer of bacterial-resistance genes in transgenic plants. [ABSTRACT FROM AUTHOR]

Abstract   Populus serves as a model tree for biotechnology and molecular biology research due to the availability of the reference genome sequence of Populus trichocarpa (Torr. & Gray) genotype ‘Nisqually-1’. However, ‘Nisqually-1’ has been shown to be very recalcitrant to micropropagation, regeneration and transformation. In this study, a highly efficient micropropagation protocol from greenhouse-grown shoot tips of ‘Nisqually-1’ was established. The optimal micropropagation protocol involves growing in vitro shoots in plant growth regulator-free Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 0.3% Gelrite® and 5–10 g L−1 of activated charcoal. Plants grown on this medium were significantly longer, and contained significantly higher concentrations of chlorophyll. This highly effective protocol provides a consistent supply of quality leaf and stem materials throughout the year for transformation experiments and other in vitro manipulations, therefore eliminating inconsistency due to seasonal and greenhouse environmental variations and the need for repetitive tissue sterilization. [ABSTRACT FROM AUTHOR]

Recent advances in Populus genomics have greatly expanded its popularity as a reference for fundamental as well as applied research in woody plants. In this review, we provide an overview of the state-of-the-science in Populus genomics research, including experimental and computational genomics. We have surveyed and summarized the following: 1) pioneering as well as more recent reports of genetics- and genomics-based investigations in Populus, 2) the positive impact of technological improvements, 3) findings from phylogenetic analyses of gene families, and 4) genomic databases. In the area of Populus experimental genomics, genetic approaches have been advanced to the genome scale with resolution to the gene and/or single nucleotide level. On the other hand, the modern “omics” approaches have been successfully applied to analysis of gene function, such as transcriptome profiling using microarrays as well as the next-generation DNA sequencing technology, and characterization of proteome and metabolome using modern instruments. In the area of Populus computational genomics, significant progresses have been made in sequence-based discovery of predicted gene function, comparative analysis of gene families, development of genomic databases, and studies of the evolutionary dynamics at both the gene and genome level. While significant advancements have been achieved in Populus genome-based science, several challenges need to be addressed, such as 1) better annotation of the Populus genome, 2) robust technology for large-scale molecular profiling, 3) efficient system for genome-wide mutagenesis, and 4) high-performance computational pipelines to keep up with the pace of the rapid accumulation of data and to integrate “omics” data into functional systems biology platforms. [ABSTRACT FROM AUTHOR]

Abstract  COBRA proteins have been shown to be involved in both cell wall expansion and/or cellulose deposition. In this paper, we analyzed all 18 COBRA genes (PtCOBRA) from the completely sequenced Populus trichocarpa genome. The 14-member PtCOBRA subfamily I proteins have high similarities to the Arabidopsis (At) COB subfamily, and members with full length sequences were predicted to possess significant potentials for a GPI-anchor site. The 4-member PtCOBRA subfamily II proteins are 45% longer than subfamily I proteins and lack ω-attachment sites at the C terminus, and are more similar to AtCOBL7 subfamily. The expression of the Populus COBRA family genes were regulated in a tissue-specific manner, and were shown to also respond differentially to inductions of hormones and environmental stimuli which affect plant cell expansion. The high levels of expressions, particularly in shoot tip and young root organs, suggests that at least some Populus COBRA genes are likely involved in regulating cell expansion. [ABSTRACT FROM AUTHOR]

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene structure, expression and function. Modified genes and consequently protein/enzymes can bridge genomics and proteomics research or facilitate commercial applications of gene and protein technologies. In this review, we will summarize various strategies, designing softwares and error correction methods for chemical gene synthesis, particularly for the synthesis and assembly of long DNA molecules based on polymerase cycling assembly. Also, we will briefly discuss some of the major applications of chemical synthesis of DNA sequences in basic research and applied areas. [ABSTRACT FROM AUTHOR]

Mannans are widespread hemicellulosic polysaccharides in plant cell walls. Hydrolysis of the internal β-1,4- d-mannopyranosyl linkage in the backbone of mannans is catalyzed by endo-β-mannanase. Plant endo-β-mannanase has been well studied for its function in seed germination. Its involvement in other plant biological processes, however, remains poorly characterized or elusive. The completed genome sequences of Arabidopsis ( Arabidopsis thaliana), rice ( Oryza sativa), and poplar ( Populus trichocarpa) provide an opportunity to conduct comparative genomic analysis of endo- β-mannanase genes in these three species. In silico sequence analysis led to the identification of eight, nine and 11 endo-β-mannanase genes in the genomes of Arabidopsis, rice, and poplar, respectively. Sequence comparisons revealed the conserved amino acids and motifs that are critical for the active site of endo-β-mannanases. Intron/exon structure analysis in conjunction with phylogenetic analysis implied that both intron gain and intron loss has played roles in the evolution of endo-β-mannanase genes. The phylogenetic analysis that included the endo-β-mannanases from plants and other organisms implied that plant endo-β-mannanases have an ancient evolutionary origin. Comprehensive expression analysis of all Arabidopsis and rice endo- β-mannanase genes showed divergent expression patterns of individual genes, suggesting that the enzymes encoded by these genes, while carrying out the same biochemical reaction, are involved in diverse biological processes. [ABSTRACT FROM AUTHOR]

It is widely accepted that gene duplication is a primary source of genetic novelty. However, the evolutionary fate of duplicated genes remains largely unresolved. The classical Ohno's Duplication-Retention-Non/Neofunctionalization theory, and the recently proposed alternatives such as subfunctionalization or duplication-degeneration-complementation, and subneofunctionalization, each can explain one or more aspects of gene fate after duplication. Duplicated genes are also affected by epigenetic changes. We constructed a phylogenetic tree using Dof (DNA binding with one finger) protein sequences from poplar (Populus trichocarpa) Torr. & Gray ex Brayshaw, Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). From the phylogenetic tree, we identified 27 pairs of paralogous Dof genes in the terminal nodes. Analysis of protein motif structure of the Dof paralogs and their ancestors revealed six different gene fates after gene duplication. Differential protein methylation was revealed between a pair of duplicated poplar Dof genes, which have identical motif structure and similar expression pattern, indicating that epigenetics is involved in evolution. Analysis of reverse transcription-PCR, massively parallel signature sequencing, and microarray data revealed that the paralogs differ in expression pattern. Furthermore, analysis of non- synonymous and synonymous substitution rates indicated that divergence of the duplicated genes was driven by positive selection. About one-half of the motifs in Dof proteins were shared by non-Dof proteins in the three plants species, indicating that motif co-option may be one of the forces driving gene diversification. We provided evidence that the Ohno's Duplication- Retention-Non/Neofunctionalization, subfunctionalization/duplication-degeneration-complementation, and subneofunctionalization hypotheses are complementary with, not alternative to, each other. [ABSTRACT FROM AUTHOR]

Vegetative propagation by cuttings is critical to the horticultural industry, particularly the ornamental and fruit sections. This article briefly reviews the traditional approaches to improve root formation, specifically with hardwood cuttings. The focus of this review has been placed on the nontraditional strategies, including infection with Agrobacterium rhizogenes and genetic engineering with rol genes, or rolB gene alone from A. rhizogenes and iaaM gene from auxin biosynthesis genes (iaaM, iaaH) from A. tumefaciens. Recent results with these new strategies have shown great potential in enhancing adventitious root formation in difficult-to-root woody species. The future perspective in understanding adventitious rooting mechanisms and new strategies for improving rooting are also discussed. doi:10.1300/J411v17n01_07 [ABSTRACT FROM PUBLISHER]

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems. [ABSTRACT FROM AUTHOR]

Abstract Agrobacterium tumefaciens was used to deliver the vhb gene into cabbage (Brassica oleracea var. Cabitata) cv. Xiaguangs parent line, 103. Using hypocotyls and cotyledon petioles as explants for infection, we obtained a transformation efficiency of 35% based on the number of transgenic shoots produced from the number of explants used for infection. Molecular analysis indicated that the vhb gene was stably integrated into the cabbage genome and that the vhb gene was expressed at the RNA level. Characterization of the vhb over-expressing transgenic plants revealed that transgenic seeds germinated faster than the wildtype controls. More importantly, the transgenic plants showed tolerance to a prolonged submergence treatment, suggesting that the vhb gene may provide an excellent tool for creation of submergence/flooding-tolerant cultivars of agriculturally important crops. [ABSTRACT FROM AUTHOR]

A putative transcription factor, named RAMY, that binds to the 20-bp O2S sequences of the regulatory region of the Amy2 gene promoter has been identified using the yeast one-hybrid system from a rice library. The full length RAMY cDNA clone encodes a 218-amino acid protein and is homologous to the late embryogenesis-abundant protein (LEA5). In vitro mutagenesis and electrophoretic mobility shift assays confirmed that RAMY can bind with O2S specifically through an unusual zinc finger with a CXCX4CX2H consensus sequence. Low levels of RAMY mRNAs were detected in rice leaves and roots by Northern blot hybridization. The plant hormone gibberellin (GA) induces expression of both RAMY and Amy2 genes, as performed by Northern blot hybridization, but the increase in RAMY mRNA level occurs prior to that of the Amy2 mRNA level in the GA-treated aleurone tissues. These data suggest that RAMY may act as a trans-acting protein and is probably involved in the GA-induced expression of the rice α-amylase gene. [ABSTRACT FROM AUTHOR]

An elite aspen hybrid (Populus×canescens×P. grandidentata) was transformed with Agrobacterium tumefaciens strain EHA105 that harbored a binary vector (pBI121) carrying the nptII gene under the nos promoter and tandem rolB-uidA (GUS) genes with the CaMV 35S or heat shock promoter. Among 32 independent kanamycin-resistant plants, 25 plants were confirmed by polymerase chain reaction and Southern blot analyses to contain all three genes, whereas five plants contained only nptII or/and uidA genes and two plants had both the rolB and nptII or uidA genes. Integration of the rolB gene significantly increased rooting ability of hardwood cuttings. Heat shock-rolB-transformed plants rooted at significantly higher percentage than the CaMV 35S-rolB-transformed plants. Heat shock treatment further enhanced rooting of heat shock-rolB-transformed plants. Exposure to exogenous auxin did not significantly increase the rooting percentage of transgenic hardwood cuttings, but increased the number of roots induced. This research shows great potential to improve rooting of hardwood cuttings of difficult-to-root woody plants which are commercially important to the horticultural and forestry industry. The transgenic plants with gain-of-function in hardwood-cutting rooting can facilitate research in the understanding of adventitious rooting from hardwood cuttings of recalcitrant woody plants. [ABSTRACT FROM AUTHOR]

The article discusses the concept of Populus as an entire living community that contains a mega-genome from both biotic organisms and abiotic environment. It provides an overview of Populus community genomics and its interactions with biotic organisms and the environment. Moreover, it outlines the applications of various classical technologies toward advancing functional genomics and Populus genetics.

To achieve a thermostable β-glucuronidase (GUS) and identify key mutation sites, we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene, gus-tr, after four rounds of DNA shuffling and screening. This variant had mutations in 15 nucleic acid sites, resulting in changes in 12 amino acids (AAs). Using gus-tr as the template, we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein. We found that six sites (Q493R, T509A, M532T, N550S, G559S and N566S) present in GUS-TR3337, were the key AAs needed to confer its high thermostability. Of these, Q493R and T509A were not reported previously as important residues for thermostability of GUS. Furthermore, all of these six mutations must be present concurrently to confer the high thermostability. We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations. Compared with the wild-type protein which lost its activity completely after 10 min at 70°C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80°C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100°C for 30 min on nitrocellulose filter. The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications. [ABSTRACT FROM AUTHOR]