Limits...
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.

Show MeSH

Related in: MedlinePlus

PCR amplification of Ara h 1 isoforms on 1% agarose gel. Lane 1: DNA size standard. Lanes 2-5: primers 1306/1307 amplify both isoforms of Ara h 1. Lanes 6-9: primers 1306/1308 amplify only Ara h 1.01. Lanes 10-13: primers 1306/1309 amplify only Ara h 1.02. GG = A. hypogaea cv. Georgia Green, Ad = A. duranensis (A genome), Ai = A. ipaensis (B genome), -ve = negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113929&req=5

Figure 1: PCR amplification of Ara h 1 isoforms on 1% agarose gel. Lane 1: DNA size standard. Lanes 2-5: primers 1306/1307 amplify both isoforms of Ara h 1. Lanes 6-9: primers 1306/1308 amplify only Ara h 1.01. Lanes 10-13: primers 1306/1309 amplify only Ara h 1.02. GG = A. hypogaea cv. Georgia Green, Ad = A. duranensis (A genome), Ai = A. ipaensis (B genome), -ve = negative control.

Mentions: Prior to designing PCR primers for Ara h 1, two genomic clones of Ara h 1 were found in GenBank. The first accession [GenBank: AF432231] was reported by Viquez et al. [17] and is identical to the cDNA sequence of accession L34402 whose encoded protein is designated Ara h 1.0101 by IUIS [2] (isoform Ara h 1.01). A second genomic clone [GenBank: AY581852] was reported by Li et al. [18] and is nearly identical to accession L38853 whose protein is referred to by Chassaigne et al. [19] as isoform 2. For clarity we will refer to this isoform as Ara h 1.02 even though this is not an official IUIS designation. PCR amplification using primers 1306 and 1307 (Table 2) produced two PCR products appearing as a doublet on agarose gel (2,241 bp for Ara h 1.01, and 2,031 bp for Ara h 1.02; Figure 1). Amplicons from gene-specific PCR were 2,211 bp for Ara h 1.01 and 1,666 bp for Ara h 1.02 (Figure 1; Table 1). Analysis of Ara h 1 PCR products from A. hypogaea and its diploid progenitors showed the presence of both genes in A. hypogaea, but only one copy in each diploid. The primer pair specific to Ara h 1.01 (1306/1308; Table 2) amplified only in A. hypogaea and A. ipaensis (B genome), while the primer pair specific to Ara h 1.02 (1306/1309; Table 2) amplified only in A. hypogaea and A. duranensis (A genome; Figure 1). Using the known sequence information, Southern blot analysis of genomic DNA from A. hypogaea was carried out to confirm that no additional copies of Ara h 1 are present in the peanut genome. Genomic DNA digested with HindIII, which has no cut sites within either gene, yielded two nearly overlapping fragments of approximately 6.5 kb each when probed with a full-length Ara h 1.01 probe (PCR product of primers 1306/1308). DNA was also digested with EcoRI, which has one cut site in each copy of Ara h 1. Southern blot analysis revealed four fragments, two from each homeolog, as expected. Lastly, the DNA was cut with AseI, which cuts Ara h 1.01 (two adjacent cut sites within the second intron), but not Ara h 1.02. As expected, three fragments were produced (Figure 2). EcoRI-digested plasmids carrying either Ara h 1.01 or Ara h 1.02 were also loaded as controls; the probe recognized both copies of the gene (data not shown).


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)

PCR amplification of Ara h 1 isoforms on 1% agarose gel. Lane 1: DNA size standard. Lanes 2-5: primers 1306/1307 amplify both isoforms of Ara h 1. Lanes 6-9: primers 1306/1308 amplify only Ara h 1.01. Lanes 10-13: primers 1306/1309 amplify only Ara h 1.02. GG = A. hypogaea cv. Georgia Green, Ad = A. duranensis (A genome), Ai = A. ipaensis (B genome), -ve = negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: PCR amplification of Ara h 1 isoforms on 1% agarose gel. Lane 1: DNA size standard. Lanes 2-5: primers 1306/1307 amplify both isoforms of Ara h 1. Lanes 6-9: primers 1306/1308 amplify only Ara h 1.01. Lanes 10-13: primers 1306/1309 amplify only Ara h 1.02. GG = A. hypogaea cv. Georgia Green, Ad = A. duranensis (A genome), Ai = A. ipaensis (B genome), -ve = negative control.
Mentions: Prior to designing PCR primers for Ara h 1, two genomic clones of Ara h 1 were found in GenBank. The first accession [GenBank: AF432231] was reported by Viquez et al. [17] and is identical to the cDNA sequence of accession L34402 whose encoded protein is designated Ara h 1.0101 by IUIS [2] (isoform Ara h 1.01). A second genomic clone [GenBank: AY581852] was reported by Li et al. [18] and is nearly identical to accession L38853 whose protein is referred to by Chassaigne et al. [19] as isoform 2. For clarity we will refer to this isoform as Ara h 1.02 even though this is not an official IUIS designation. PCR amplification using primers 1306 and 1307 (Table 2) produced two PCR products appearing as a doublet on agarose gel (2,241 bp for Ara h 1.01, and 2,031 bp for Ara h 1.02; Figure 1). Amplicons from gene-specific PCR were 2,211 bp for Ara h 1.01 and 1,666 bp for Ara h 1.02 (Figure 1; Table 1). Analysis of Ara h 1 PCR products from A. hypogaea and its diploid progenitors showed the presence of both genes in A. hypogaea, but only one copy in each diploid. The primer pair specific to Ara h 1.01 (1306/1308; Table 2) amplified only in A. hypogaea and A. ipaensis (B genome), while the primer pair specific to Ara h 1.02 (1306/1309; Table 2) amplified only in A. hypogaea and A. duranensis (A genome; Figure 1). Using the known sequence information, Southern blot analysis of genomic DNA from A. hypogaea was carried out to confirm that no additional copies of Ara h 1 are present in the peanut genome. Genomic DNA digested with HindIII, which has no cut sites within either gene, yielded two nearly overlapping fragments of approximately 6.5 kb each when probed with a full-length Ara h 1.01 probe (PCR product of primers 1306/1308). DNA was also digested with EcoRI, which has one cut site in each copy of Ara h 1. Southern blot analysis revealed four fragments, two from each homeolog, as expected. Lastly, the DNA was cut with AseI, which cuts Ara h 1.01 (two adjacent cut sites within the second intron), but not Ara h 1.02. As expected, three fragments were produced (Figure 2). EcoRI-digested plasmids carrying either Ara h 1.01 or Ara h 1.02 were also loaded as controls; the probe recognized both copies of the gene (data not shown).

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