Developing a 2-Phase Screening System to Assess the in vitro and in vivo Stability of Engineering Native Seed Storage Proteins

Modification of the composition of grain storage proteins is an intensively developing area of plant biotechnology, which is of particular importance for sorghum – high-yielding drought tolerant crop. Compared to other cereals, the majority of sorghum cultivars and hybrids are characterized by reduced nutritional value that is caused by a low content of essential amino acids in the seed storage proteins (kafirins), and resistance of kafirins to protease digestion. RNA interference (RNAi) by suppressing synthesis of individual kafirin subclasses may be an effective approach to solve this problem. In this chapter, we review published reports on RNAi silencing of the kafirin-encoding genes. In addition, we present new experimental data on phenotypic effects of RNAi-silencing of γ-KAFIRIN-1 gene in sorghum cv. Avans. To obtain RNAi mutants with γ-KAFIRIN-1 gene silencing we used Agrobacterium-mediated genetic transformation. Transgenic kernels had modified endosperm type with reduced vitreous layer and significantly improved in vitro protein digestibility (93% vs. 57%, according to the densitometry of SDS-PAGE patterns). SDS-PAGE of transgenic kernels showed lowered level of kafirins and appearance of globulin proteins, which were not observed in the original cultivar. For the first time, the cases of instability of inserted genetic construct were identified: elimination of ubi1-intron that is a constituent part of the genetic construct for RNAi silencing, or nos-promotor governing expression of the marker gene (bar) (in the RNAi mutants of cv. Zheltozernoe 10). The research findings presented in this chapter provide strong evidence that RNA interference can be used for improvement of the nutritional properties of sorghum grain.

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Seed Storage Proteins, 2S Albumin And 11S Globulin, Associated to Severe Allergic Reactions after Flaxseed Intake

Journal of Investigational Allergology and Clinical Immunology ◽

10.18176/jiaci.0713 ◽

2021 ◽

Vol 32 (5) ◽

Author(s):

C Bueno-Díaz ◽

C Biserni ◽

L Martín-Pedraza ◽

M de las Heras ◽

C Blanco ◽

...

Keyword(s):

Mass Spectrometry ◽

Storage Proteins ◽

Binding Capacity ◽

Seed Storage ◽

Seed Storage Proteins ◽

Cross Reactivity ◽

2S Albumin ◽

11S Globulin ◽

Flaxseed Extract

Background: Given the increased popularity of flaxseed in meals, several cases of allergy to these seeds have been reported. Little is known about allergens implicated in hypersensitivity reactions to these seeds. The present work aimed to identify the allergens involved in IgE-mediated reactions in five patients with a clinical history of severe systemic symptoms after flaxseed consumption. Methods: Proteins susceptible to be allergens with IgE-binding capacity were purified from flaxseed extract by chromatographic techniques. Their identification was achieved via MALDI-TOF mass spectrometry. Immunoassays were performed using the five allergic patient’s era either by testing them individually or as a pool. Results: Four out of five patients recognized a low-molecular-mass protein (around 13kDa) by immunoblotting of the flaxseed extract, while two patients recognized a protein of approximately 55 kDa. They were identified by mass spectrometry as flaxseed 2S albumin, included into WHO/IUIS allergen nomenclature as Lin u 1,and 11S globulin, respectively. Inhibition assays revealed in vitro IgE-cross-reactivity of Lin u 1 with peanut and cashew nut proteins, while IgE recognition of 11S globulin by patients’ sera was partially inhibited by several plant-derived sources. Conclusions: Seed storage proteins from flaxseed were involved in the development of severe symptoms in five individuals and exhibited cross-reactivity with other allergenic sources. Besides the severity of flaxseed allergy in patients sensitized to 2S albumin, it is the first time that the 11S globulin is identified as a potential allergen. We consider that these data should be taken into account for a more accurate diagnosis of patients.

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