Gly m 5/Gly m 8 fusion component as a potential novel candidate molecule for diagnosing soya bean allergy in Japanese children

Maruyama, Nobuyuki; Sato, Sakura; Cabanos, Cerrone; Ito, Komei; Ebisawa, Motohiro

doi:10.1111/cea.13231

Cited by 16 publications

(25 citation statements)

References 28 publications

(71 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Membranes were blocked with 5% skim milk (Wako) overnight at 4°C. Recombinant Gly m Bd 30 k was prepared using the baculovirus expression system as described in [5]. Using the pET52 vector (Merck-Millipore, Burlington, USA), His 10 -tagged Gly m Bd 28 K was expressed in Escherichia coli BL21(DE3).…”

Section: Protein Analyses In Mature Seedsmentioning

confidence: 99%

“…In the USA and Europe, 5 to 8% of babies and 2% of adults are allergic to soybean [4]. Several subunits of major storage proteins such as 7S and 11S globulins and 2S albumin are representative soybean allergens [5]. The vicilin-like glycoprotein Gly m Bd 28 K and the oilbody-associated protein Gly m Bd 30 K are also reported as major soybean allergens [6,7].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

et al. 2020

Self Cite

View full text Add to dashboard Cite

Background Soybean (Glycine max) is a major protein crop, because soybean protein has an amino acid score comparable to that of beef and egg white. However, many allergens have been identified among soybean proteins. A decrease in allergenic protein levels would be useful for expanding the market for soybean proteins and processed foods. Recently, the CRISPR/Cas9 system has been adopted as a powerful tool for the site-directed mutagenesis in higher plants. This system is expected to generate hypoallergenic soybean varieties. Results We used two guide RNAs (gRNAs) and Agrobacterium-mediated transformation for simultaneous site-directed mutagenesis of two genes encoding the major allergens Gly m Bd 28 K and Gly m Bd 30 K in two Japanese soybean varieties, Enrei and Kariyutaka. We obtained two independent T0 Enrei plants and nine T0 Kariyutaka plants. Cleaved amplified polymorphic sequence (CAPS) analysis revealed that mutations were induced in both targeted loci of both soybean varieties. Sequencing analysis showed that deletions were the predominant mutation type in the targeted loci. The Cas9-free plants carrying the mutant alleles of the targeted loci with the transgenes excluded by genetic segregation were obtained in the T2 and T3 generations. Variable mutational spectra were observed in the targeted loci even in T2 and T3 progenies of the same T0 plant. Induction of multiple mutant alleles resulted in six haplotypes in the Cas9-free mutants derived from one T0 plant. Immunoblot analysis revealed that no Gly m Bd 28 K or Gly m Bd 30 K protein accumulated in the seeds of the Cas9-free plants. Whole-genome sequencing confirmed that a Cas9-free mutant had also no the other foreign DNA from the binary vector. Our results demonstrate the applicability of the CRISPR/Cas9 system for the production of hypoallergenic soybean plants. Conclusions Simultaneous site-directed mutagenesis by the CRISPR/Cas9 system removed two major allergenic proteins from mature soybean seeds. This system enables rapid and efficient modification of seed components in soybean varieties.

show abstract

Section: Protein Analyses In Mature Seedsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…Membranes were blocked with 5% skim milk (Wako, Osaka, Japan) overnight at 4°C. Recombinant Gly m Bd 30K protein was produced using the baculovirus expression system as described in Maruyama et al (2018).…”

Section: Protein Analyses Of Mature Seedsmentioning

confidence: 99%

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

et al. 2021

Self Cite

View full text Add to dashboard Cite

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables.Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence (CAPS) analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

show abstract

“…Allergies to foods have been associated with severe asthma exacerbations while asthma seems to be a risk factor for life-threatening food induced anaphylaxis. Novel allergens have also been identified for orange, 63 donkey's milk 64 and soya 65 in 2018.…”

Section: Asthma and Food Allergymentioning

confidence: 99%

Developments in the field of clinical allergy in 2018 through the eyes of Clinical and Experimental Allergy, Part II

Roberts

Almqvist

Boyle

et al. 2019

Clin Experimental Allergy

View full text Add to dashboard Cite

show abstract

Gly m 5/Gly m 8 fusion component as a potential novel candidate molecule for diagnosing soya bean allergy in Japanese children

Abstract: A fusion protein of Gly m 8 and the extension region of α' subunit of Gly m 5 could potentially diagnose soya bean allergy in paediatric patients. Fusion proteins may be useful for producing novel allergen components with improved diagnostic value.

Cited by 16 publications

References 28 publications

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation

Developments in the field of clinical allergy in 2018 through the eyes of Clinical and Experimental Allergy, Part II

Contact Info

Product

Resources

About