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

Knoll JE, Ramos ML, Zeng Y, Holbrook CC, Chow M, Chen S, Maleki S, Bhattacharya A, Ozias-Akins P - BMC Plant Biol. (2011)

Bottom Line: Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses.A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein.This work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticulture/NESPAL, University of Georgia-Tifton Campus, Tifton, GA 31793, USA.

ABSTRACT

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.

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Analysis of seed protein extracts from Ara h 2.02 knockout mutant. A - Coomassie blue stained SDS-PAGE of seed protein extracts, with equal amounts of total protein loaded in each lane. Lane wt: wild type (Tifrunner). Lane 1: homozygous mutant. Lane 2: heterozygote. B - Western blot of seed protein extracts using anti-Ara h 2 antibodies, which recognize both isoforms of the allergen. Antibodies also recognize Ara h 6. Lane wt: wild type (Tifrunner). Lane 1: homozygous mutant. Lane 2: heterozygote. C - 2D DIGE analysis of seed protein extracts from wild-type (Tifrunner) labeled with Cy3 (green) and Ara h 2.02 knockout mutant labeled with Cy2 (red). The white box denotes the four spots representing Ara h 2 isoforms.
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Figure 4: Analysis of seed protein extracts from Ara h 2.02 knockout mutant. A - Coomassie blue stained SDS-PAGE of seed protein extracts, with equal amounts of total protein loaded in each lane. Lane wt: wild type (Tifrunner). Lane 1: homozygous mutant. Lane 2: heterozygote. B - Western blot of seed protein extracts using anti-Ara h 2 antibodies, which recognize both isoforms of the allergen. Antibodies also recognize Ara h 6. Lane wt: wild type (Tifrunner). Lane 1: homozygous mutant. Lane 2: heterozygote. C - 2D DIGE analysis of seed protein extracts from wild-type (Tifrunner) labeled with Cy3 (green) and Ara h 2.02 knockout mutant labeled with Cy2 (red). The white box denotes the four spots representing Ara h 2 isoforms.

Mentions: Lastly, a G to A transition was identified in the start codon of Ara h 2.02. A downstream ATG is out of frame, and so a protein knockout was expected. Two M3 seeds were recovered, a small chip was taken from each for protein analysis, and the seeds were planted. Both seeds grew into phenotypically normal plants. SDS-PAGE analysis of the seed protein extracts confirmed that one of the seeds was indeed missing the 21 kD band which represents the Ara h 2.02 protein [9], and was thus homozygous for the mutation (Figure 4A). The other seed appeared to have a reduced quantity of Ara h 2.02; DNA sequence analysis (data not shown) confirmed that this plant was a heterozygote. Western blot analysis (Figure 4B) also confirmed the absence of Ara h 2.02 protein in the homozygous mutant. Further analysis with 2-D difference gel electrophoresis (2-D DIGE) confirmed that both of the Ara h 2.02 isoforms, shown to differ by a two amino acid truncation at the carboxy terminus [24], were missing in the homozygous mutant line (Figure 4C).


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

Knoll JE, Ramos ML, Zeng Y, Holbrook CC, Chow M, Chen S, Maleki S, Bhattacharya A, Ozias-Akins P - BMC Plant Biol. (2011)

Analysis of seed protein extracts from Ara h 2.02 knockout mutant. A - Coomassie blue stained SDS-PAGE of seed protein extracts, with equal amounts of total protein loaded in each lane. Lane wt: wild type (Tifrunner). Lane 1: homozygous mutant. Lane 2: heterozygote. B - Western blot of seed protein extracts using anti-Ara h 2 antibodies, which recognize both isoforms of the allergen. Antibodies also recognize Ara h 6. Lane wt: wild type (Tifrunner). Lane 1: homozygous mutant. Lane 2: heterozygote. C - 2D DIGE analysis of seed protein extracts from wild-type (Tifrunner) labeled with Cy3 (green) and Ara h 2.02 knockout mutant labeled with Cy2 (red). The white box denotes the four spots representing Ara h 2 isoforms.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113929&req=5

Figure 4: Analysis of seed protein extracts from Ara h 2.02 knockout mutant. A - Coomassie blue stained SDS-PAGE of seed protein extracts, with equal amounts of total protein loaded in each lane. Lane wt: wild type (Tifrunner). Lane 1: homozygous mutant. Lane 2: heterozygote. B - Western blot of seed protein extracts using anti-Ara h 2 antibodies, which recognize both isoforms of the allergen. Antibodies also recognize Ara h 6. Lane wt: wild type (Tifrunner). Lane 1: homozygous mutant. Lane 2: heterozygote. C - 2D DIGE analysis of seed protein extracts from wild-type (Tifrunner) labeled with Cy3 (green) and Ara h 2.02 knockout mutant labeled with Cy2 (red). The white box denotes the four spots representing Ara h 2 isoforms.
Mentions: Lastly, a G to A transition was identified in the start codon of Ara h 2.02. A downstream ATG is out of frame, and so a protein knockout was expected. Two M3 seeds were recovered, a small chip was taken from each for protein analysis, and the seeds were planted. Both seeds grew into phenotypically normal plants. SDS-PAGE analysis of the seed protein extracts confirmed that one of the seeds was indeed missing the 21 kD band which represents the Ara h 2.02 protein [9], and was thus homozygous for the mutation (Figure 4A). The other seed appeared to have a reduced quantity of Ara h 2.02; DNA sequence analysis (data not shown) confirmed that this plant was a heterozygote. Western blot analysis (Figure 4B) also confirmed the absence of Ara h 2.02 protein in the homozygous mutant. Further analysis with 2-D difference gel electrophoresis (2-D DIGE) confirmed that both of the Ara h 2.02 isoforms, shown to differ by a two amino acid truncation at the carboxy terminus [24], were missing in the homozygous mutant line (Figure 4C).

Bottom Line: Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses.A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein.This work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticulture/NESPAL, University of Georgia-Tifton Campus, Tifton, GA 31793, USA.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus