TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.)

Knoll, Joseph E.; Ramos, M. Laura; Zeng, Yajuan; Holbrook, C. Corley; Chow, Marjorie; Chen, Sixue; Maleki, Soheila; Bhattacharya, Anjanabha; Ozias-Akins, Peggy
January 2011
BMC Plant Biology;2011, Vol. 11 Issue 1, p81
Academic Journal
Background: Allergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut. Results: Two similar copies of a major allergen gene, Ara h 1, have been identified in tetraploid peanut, one in each subgenome. The same situation has been shown for major allergen Ara h 2. Due to the challenge of discriminating between homeologous genes in allotetraploid peanut, nested PCR was employed, in which both gene copies were amplified using unlabeled primers. This was followed by a second PCR using gene-specific labeled primers, heteroduplex formation, CEL1 nuclease digestion, and electrophoretic detection of labeled fragments. Using ethyl methanesulfonate (EMS) as a mutagen, a mutation frequency of 1 SNP/967 kb (3,420 M2 individuals screened) was observed. The most significant mutations identified were a disrupted start codon in Ara h 2.02 and a premature stop codon in Ara h 1.02. Homozygous individuals were recovered in succeeding generations for each of these mutations, and elimination of Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses. TILLING also was used to identify mutations in fatty acid desaturase AhFAD2 (also present in two copies), a gene which controls the ratio of oleic to linoleic acid in the seed. A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein. A mutation in AhFAD2A was predicted to restore function to the normally inactive enzyme. Conclusions: This work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity. TILLING in peanut can be extended to virtually any gene, and could be used to modify other traits such as nutritional properties of the seed, as shown in this study.


Related Articles

  • Regulation of Rubisco activase and its interaction with Rubisco. Portis, Archie R.; Cishan Li; Dafu Wang; Salvucci, Michael E. // Journal of Experimental Botany;May2008, Vol. 59 Issue 7, p1597 

    The large, α-isoform of Rubisco activase confers redox regulation of the ATP/ADP response of the ATP hydrolysis and Rubisco activation activities of the multimeric activase holoenzyme complex. The α-isoform has a C-terminal extension that contains the redox-sensitive cysteine residues and...

  • Role of the Calcium-Binding Residues Asp231, Asp233, and Asp438 in Alpha-Amylase of Bacillus amyloliquefaciens as Revealed by Mutational Analysis. Yang Liu; Wei Shen; Gui-yang Shi; Zheng-xiang Wang // Current Microbiology;Mar2010, Vol. 60 Issue 3, p162 

    Role of the calcium-binding residues Asp231, Asp233, and Asp438 of Bacillus amyloliquefaciens α-amylase (BAA) on the enzyme properties was investigated by site-directed mutagenesis. The calcium-binding residues Asp231, Asp233, and Asp438 were replaced with Asn, Asn, and Gly to produce the...

  • Design of phosphodiesterase 4D (PDE4D) allosteric modulators for enhancing cognition with improved safety. Burgin, Alex B.; Magnusson, Olafur T.; Singh, Jasbir; Witte, Pam; Staker, Bart L.; Bjornsson, Jon M.; Thorsteinsdottir, Margret; Hrafnsdottir, Sigrun; Hagen, Timothy; Kiselyov, Alex S.; Stewart, Lance J.; Gurney, Mark E. // Nature Biotechnology;Jan2010, Vol. 28 Issue 1, p63 

    Phosphodiesterase 4 (PDE4), the primary cAMP-hydrolyzing enzyme in cells, is a promising drug target for a wide range of conditions. Here we present seven co-crystal structures of PDE4 and bound inhibitors that show the regulatory domain closed across the active site, thereby revealing the...

  • Characterization of hemizygous deletions in Citrus using array-Comparative Genomic Hybridization and microsynteny comparisons with the poplar genome. Ríos, Gabino; Naranjo, Miguel A.; Iglesias, Domingo J.; Ruiz-Rivero, Omar; Geraud, Marion; Usach, Antonio; Talón, Manuel // BMC Genomics;2008, Vol. 9, Special section p1 

    Background: Many fruit-tree species, including relevant Citrus spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and...

  • Prediction of Enzyme Mutant Activity Using Computational Mutagenesis and Incremental Transduction. Basit, Nada; Wechsler, Harry // Advances in Bioinformatics;2011, p1 

    Wet laboratory mutagenesis to determine enzyme activity changes is expensive and time consuming. This paper expands on standard one-shot learning by proposing an incremental transductive method (T2bRF) for the prediction of enzyme mutant activity duringmutagenesis using Delaunay tessellation and...

  • Expression, Purification, Biochemical and Pharmacological Characterization of a Recombinant Aprotinin Variant. Apeler, Heiner; Peters, Jorg; Schr�der, Werner; Schneider, Karl-Heinz; Lemm, Georg; Hinz, Vower; Rossouw, Gawle J.; Dembowsky, Klaus // Drug Research / Arzneimittel-Forschung;Aug2004, Vol. 54 Issue 8, p483 

    Aprotinin (CAS 9087-70-1) is known as a potent inhibitor of serine proteases such as trypsin, plasmin, tissue and plasma kallikrein. In this study, an aprotinin variant was designed by means of rationale mutagenesis that differs from aprotinin by two amino acids in the active site and by seven...

  • Improvement of the activity of arylmalonate decarboxylase by random mutagenesis. Terao, Y.; Miyamoto, K.; Ohta, H. // Applied Microbiology & Biotechnology;Dec2006, Vol. 73 Issue 3, p647 

    Arylmalonate decarboxylase (EC catalyzes enantioselective decarboxylation of α-aryl-α-methylmalonates to give optically pure α-arylpropionates. Recently, we have succeeded in creating a double mutant enzyme that gave opposite enantionmer as the product. Unfortunately, however,...

  • A Paenibacillus sp. dextranase mutant pool with improved thermostability and activity.  // Applied Microbiology & Biotechnology;Jul2007, Vol. 75 Issue 5, p1071 

    Random mutagenesis was used to create a library of chimeric dextranase (dexl) genes. A plate-screening protocol was developed with improved thermostability as a selection criterion. The mutant library was screened for active dextranase variants by observing clearing zones on dextran-blue agar...

  • Effect of Silent Mutations in Translational Initial Region on the Production of Recombinant Cutinase in Escherichia coli. Zhi-guo Liu; Li Zhu; Kong-liang Zhu; Sheng Chen; Jian Chen; Jing Wu // Current Microbiology;Apr2011, Vol. 62 Issue 4, p1302 

    Translational Initial Region (TIR) is the threshold of an intracellular translation process, and tiny alterations in this region are reported to intensely influence the downstream expression. Such property provides a potential utilization in extracellular production of recombinant enzyme. As an...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics