Search

Background: RNA interference is a very effective approach for studies on gene function and may be an efficient method for controlling pests. Trehalase is a key gene in the chitin biosynthesis pathway in insects. Five trehalase genes have been cloned in Tribolium castaneum, though it is not known whether the detailed functions of these trehalases can be targeted for pest control., Results: The functions of all five trehalase genes were studied using RNAi, and the most important results showed that the expression of all 12 genes decreased significantly from 12 to 72 h compared with the control groups, except GP1 at 72 h, when the expression of the TcTre2 gene was suppressed. The results also revealed different abnormal phenotypes, and the observed mortality rates ranged from 17 to 42 %. The qRT-PCR results showed that the expression of TPS, GS, two GP, CHS1a and CHS1b genes decreased significantly, while that of the CHS2 gene decreased or increased after RNAi after the five trehalases were silenced at 48 h. In addition, TPS gene expression decreased from 12 to 72 h after dsTcTre injection., Conclusions: These results demonstrate that silencing of any individual trehalase gene, especially Tre1-4 and Tre2 gene can lead to moulting deformities and a high mortality rate through the regulation of gene expression in the chitin biosynthesis pathway and may be a potential approach for pest control in the future.

Background: Trehalose is related to several types of stress responses, especially freezing response in baker's yeast (Saccharomyces cerevisiae). It is desirable to manipulate trehalose-related genes to create yeast strains that better tolerate freezing-thaw stress with improved fermentation capacity, which are in high demand in the baking industry., Results: The strain overexpressing MAL62 gene showed increased trehalose content and cell viability after prefermention-freezing and long-term frozen. Deletion of NTH1 in combination of MAL62 overexpression further strengthens freezing tolerance and improves the leavening ability after freezing-thaw stress., Conclusions: The mutants of the industrial baker's yeast with enhanced freezing tolerance and leavening ability in lean dough were developed by genetic engineering. These strains had excellent potential industrial applications.

Background: Trehalases have potential applications in several fields, including food additives, insecticide development, and transgenic plant. In the present study, we focused on a trehalase from the marine bacterium Zunongwangia sp., which hydrolyzes trehalose to glucose., Results: A novel gene, treZ (1590 bp) encoding an α, α-trehalase of 529 amino acids was cloned from Zunongwangia sp., and TreZ was found to have an optimal activity at 50 °C and pH 6. The activity of TreZ was increased by the presence of NaCl, showing the highest activity (136 %) at 1 M NaCl. A variant C4 with improved catalytic activity was obtained by error-prone PCR and followed by a 96-well plate high-throughput screening. The variant C4 with two altered sites (Y227H, and R442G) displayed a 3.3 fold increase in catalytic efficiency (k cat/K m, 1143.40 mmol(-1) s(-1)) compared with the wild type enzyme (265.91 mmol(-1) s(-1)). In order to explore the contribution of the mutations found in variant C4 to the increased catalytic activity, two mutants Y227H and R442G were constructed by site-directed mutagenesis. The results showed that the catalytic efficiencies of Y227H and R442G were 416.78 mmol(-1) s(-1) and 740.97 mmol(-1) s(-1), respectively, indicating that both mutations contributed to the increased catalytic efficiency of variant C4. The structure modeling and substrate docking revealed that the substitution Y227H enlarged the shape of the binding pocket, to improve the binding of the substrate and the release of the products; while the substitution R442G reduced the size of the side chain and decreased the steric hindrance, which contributed to channel the substrate into the active cavity easier and promote the release of the product., Conclusion: In this study, a novel trehalase was cloned, purified, characterized, and engineered. A variant C4 with dramatically improved catalytic activity was obtained by directed evolution, and the mutation sites Y227H and R442G were found to play a significant role in the catalytic efficiency. The overall results provide useful information about the structure and function of trehalase.

Background: The entomopathogenic fungus Metarhizium acridum has been used as an important biocontrol agent instead of insecticides for controlling crop pests throughout the world. However, its virulence varies with environmental factors, especially temperature. Neutral trehalase (Ntl) hydrolyzes trehalose, which plays a role in environmental stress response in many organisms, including M. acridum. Demonstration of a relationship between Ntl and thermotolerance or virulence may offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi through genetic engineering., Results: We selected four Ntl over-expression and four Ntl RNA interference (RNAi) transformations in which Ntl expression is different. Compared to the wild-type, Ntl mRNA expression was reduced to 35-66% in the RNAi mutants and increased by 2.5-3.5-fold in the over-expression mutants. The RNAi conidiospores exhibited less trehalase activity, accumulated more trehalose, and were much more tolerant of heat stress than the wild-type. The opposite effects were found in conidiospores of over-expression mutants compared to RNAi mutants. Furthermore, virulence was not altered in the two types of mutants compared to the wild type., Conclusions: Ntl controlled trehalose accumulation in M. acridum by degrading trehalose, and thus affected conidiospore thermotolerance. These results offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi without affecting virulence.

Background: The chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. Although insects possess two types, soluble trehalase (Tre-1) and membrane-bound trehalase (Tre-2), very little is known about Tre-2 and the difference in function between Tre-1 and Tre-2., Results: To gain an insight into trehalase functions in insects, we investigated a putative membrane-bound trehalase from Spodoptera exigua (SeTre-2) cloned from the fat body. The deduced amino acid sequence of SeTre-2 contains 645 residues and has a predicted molecular weight of approximately 74 kDa and pI of 6.01. Alignment of SeTre-2 with other insect trehalases showed that it contains two trehalase signature motifs and a putative transmembrane domain, which is an important characteristic of Tre-2. Comparison of the genomic DNA and cDNA sequences demonstrated that SeTre-2 comprises 13 exons and 12 introns. Southern blot analysis revealed that S. exigua has two trehalase genes and that SeTre-2 is a single-copy gene. Northern blot analyses showed that the SeTre-2 transcript is expressed not only in the midgut, as previously reported for Bombyx mori, but also in the fat body and Malpighian tubules, although expression patterns differed between the midgut and fat body. SeTre-2 transcripts were detected in the midgut of feeding stage larvae, but not in pupae, whereas SeTre-2 mRNA was detected in the fat body of fifth instar larvae and pupae., Conclusion: These findings provide new data on the tissue distribution, expression patterns and potential function of membrane-bound trehalase. The results suggest that the SeTre-2 gene may have different functions in the midgut and fat body.

Background: The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p., Results: In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain., Conclusions: These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.