Progress 07/01/07 to 06/30/09
Outputs
OUTPUTS: Larval stages of the western corn rootworm have an obligatory host relationship with grasses (Graminae), and although corn is believed to be the primary host plant, development on a variety of Midwestern prairie grasses has also been documented. A role for cellulose digestion in the nutritional ecology of larval rootworm development has only recently been suggested based on the identification of Expressed Sequence Tags (EST's) derived from a gut cDNA library (Siegfried et al., 2005), which exhibit a high degree of similarity with genes from other insects that have been implicated in cellulose metabolism. The initial analysis of sequence data revealed at least two different endogenous cellulases based on similarity to genes from other insect species that encode proteins with documented cellulase activity. The complete ORF of one of these genes was amplified by 3' and 5' rapid amplification of cDNA ends (RACE PCR), sequenced, and the deduced amino acid sequence was shown to exhibit high similarity to β-1,4 endoglucanases from fungi, nematodes, and other insects. Both putative cellulase sequences exhibit highly conserved residues in the active site of the enzyme. The sequence exhibits a catalytic motif characteristic of the glycohydrolase family (GHF) 45 that is the substrate binding site for this enzyme class. Importantly, we have made significant progress in cloning and expression of this gene and have begun to characterize its activity toward model substrates. A recombinant Dvv1 was obtained using the Cell-Free Expression System which allows synthesis of proteins from both circular plasmid and linear PCR templates. This system allows development of high-throughput synthesis of proteins and expression of genes toxic to in vivo systems. Using a polyclonal antiserum derived from a synthetic peptide representing 18 amino acid residues near the end N-terminus, expression of the recombinant protein was confirmed. The observed molecular weight of the recombinant protein was identical to that calculated based on the amino acid sequence. A higher molecular weight protein was also detected although its significance is still uncertain. PARTICIPANTS: Personnel Supported: Arnubio Valencia; Visiting Scientist, University of Manazales, Colombia Analiza Alves, Ph.D. Student, Department of Entomology, (Graduated August, 2008) Matthew Moore, UNL Undergraduate, Insect Science Major, UCARE program participant TARGET AUDIENCES: Biofuels industry. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Because rootworms have evolved with corn and apparently utilize corn cellulose as a source of nutrition, an understanding of the biochemical pathways that result in cellulose digestion could lead to novel sources of enzymes for cellulosic ethanol production.
Publications
- Siegfried, B.D., A. Valencia, and A.P. Alves. 2009. Molecular cloning and cell-free expression of an endogenous endoglucanase belonging to GHF45 from the western corn rootworm, Diabortica virgifera virgifera. Provisional Patent Application KSS 1044
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: I. Methods for the isolation of luminal gut proteins and measurement of endoglucanase, exoglucanase, and β-glucosidase activities from late instar western corn rootworm larvae have been established using a number of model substrates. Endoglucanase activity was measured with the substrate carboxymethyl cellulose (CMCase) and quantitatively determined by measuring the reducing sugars liberated using the dinitrosalicylic (DNS) reagent method. The beta-glucosidase activity was determined using both salicin and p-nitrophenyl-beta-D-glucoside (pNPG) as substrates, and therefore referred to as salicinase and pNPGase, respectively. The salicinase assay involved measurement of reducing sugars while the pNPGase activity was determined by measuring the production of p-nitrophenol spectrophotometrically. Exoglucanse activity was measured using p-nitrophenyl-β-D-cellobioside (p-NPC) as substrate and again involved measuring the production of p-nitrophenol. General extraction procedures of insect cellulases: For cellulase distribution studies, acetone-treated whole body larvae were divided into three regions: (i) forgut, (ii) midgut and (iii) hindgut, in an attempt to conserve the correspondent insect intestinal section. These intestinal sections were homogenized manually in ice-cold homogenization buffer. Additionally, late instar larvae were dissected under a stereo-microscope in order to obtain the complete insect intestinal tract. Endoglucanase, beta-glucosidase and exoglucanase activities in the intestinal tract of western corn rootworm were approximately evenly distributed between the foregut and midgut with much lower activities in the hindgut. The optimal pH of luminal gut extracts was determined by pre-incubating the enzymes in appropriate buffers. The effect of temperature was examined under standard conditions by varying the temperature from18-70 deg C. It was observed that endoglucanse, exoglucanase, and beta-glucosidase exhibit highest activity when the pH of the buffer solution is between 5.0 and 7.0 with a peak at about pH 6.0. The temperature optimum for all three activities was between 40-45 deg C and generally appeared to be stable at higher temperatures maintaining over 30% of residual activity at 70 deg C when compared with the temperature optimum (4 deg C). II. We have identified several gene fragments from a western corn rootworm midgut cDNA library that encode putative proteins resembling polysaccharide degrading enzymes. The initial analysis of sequence data revealed at least two different endogenous cellulases based on similarity to genes from other insect species that encode proteins with documented cellulase activity. The complete ORF of one of these genes has been amplified by 3' and 5' rapid amplification of cDNA ends (RACE PCR), sequenced, and the deduced amino acid sequence shown to exhibit high similarity to β-1,4 endoglucanases from fungi, nematodes and other insects. Both putative cellulase sequences exhibit highly conserved residues in the active site of the enzyme. The sequence contains a catalytic motif characteristic of the glycohydrolase 45 family that is the substrate binding site. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Because rootworms have evolved with corn and apparently utilize corn cellulose as a source of nutrition, an understanding of the biochemical pathways that result in cellulose digestion could lead to novel sources of enzymes for cellulosic ethanol production.
Publications
- No publications reported this period
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