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.2021 Jun 7:8:678421.
doi: 10.3389/fnut.2021.678421. eCollection 2021.

Identification and Characterization of the Seed Storage Proteins and Related Genes ofCannabis sativa L

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Identification and Characterization of the Seed Storage Proteins and Related Genes ofCannabis sativa L

Xin Sun et al. Front Nutr..

Abstract

Hemp (Cannabis sativa L.) seed is emerging as a novel source of plant protein owing to its rich protein content and reasonable nutritional structure. In the current study, the storage proteins of hemp seed were extracted using different methods. The modified Osborne method yielded maximum extraction of the hemp seed storage proteins, while degreasing had little effect on the hemp seed protein (HSP) extraction. Protein identification results revealed that 11S globulin (edestin) was the most abundant protein in hemp seed, and the molecular weights of the two subunits of this protein were ~35 and 20 kDa, respectively. The second most abundant protein was 2S albumin (Cs2S), with a molecular weight of ~14-15 kDa. The least abundant protein was 7S vicilin-like protein (Cs7S), with a molecular weight of ~47 kDa. Subsequently, gene families encoding these three storage protein classes, including three genes for edestin, two for Cs2S, and one for Cs7S, were cloned and then analyzed for amino acid composition and structure. The three edestins were different in their amino acid sequences and calculated molecular weights. The analysis of coding sequences revealed a higher percentage of similarity (62.7%) betweenEdestin1 andEdestin3, while the similarity decreased significantly to ~57% betweenEdestin1 andEdestin2, and 58% betweenEdestin2 andEdestin3. The calculated protein molecular weight was the highest for the protein encoded byEdestin1 and the smallest for the protein encoded byEdestin2. All three edestins were rich in arginine, while Edestin3 had a higher methionine content relative to that in the other two, which proved that Edestin3 had a better nutritional value. Cs2S and Cs7S were different from those reported in previous studies. Therefore, it could be inferred that amino acid composition varies with different hemp cultivars. The current research brought significant theoretical advance in illuminating the understanding of hemp seed storage protein and would have significance for future research on improving the nutritional quality of hemp seed and developing bioactive peptides.

Keywords: Cannabis sativa; albumin; edestin; seed storage protein; vicilin-like protein.

Copyright © 2021 Sun, Sun, Li, Wu and Wang.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Flowchart of the production of HSP from fresh hemp seeds using different methods. Different colors indicate the different extraction methods; the Osborne method is indicated in GREEN, alkali extraction and acid precipitation method is indicated in YELLOW, protein extraction kit method is indicated in RED, and the common operating steps are indicated in BLUE. The concentration of NaOH and NaCl solutions used was 1 M, while the concentration of the HCl solution used was 0.1 M.
Figure 2
Figure 2
SDS-PAGE results under different conditions.(A) SDS-PAGE electrophoresis of defatted/undefatted hempseed protein extracted using different methods. The time indicated the protein boiling time at 95°C and the 2ME concentration was 2.5%;(B) SDS-PAGE electrophoresis of raw hempseed protein extracted using the “acid precipitation and alkaline extraction” method and the Osborne fractionation method (for water-soluble and salt-soluble proteins, respectively). The salt-soluble protein-1 was extracted using NaCl, while the salt-soluble protein-2 was extracted using urea. The concentrations of NaCl was 1 M and of urea was 8 M, the boiling time was 5 min.
Figure 3
Figure 3
Basic statistical charts for the protein identifications.(A) Identification results for proteins in the 40–50 kDa range.(B) Identification results for proteins in the 30–40 kDa range.(C,D) Identification results for proteins in the 20 kDa range.(E) Identification results for proteins in the <20 kDa range.
Figure 4
Figure 4
The electropherogram of gene cloning.(A) Result of PCR amplification.(B,C) PCR results for the recombinant vectors; the red arrows indicate the positive clones sent for sequencing. E1: edestin1; E2: edestin2; E3: edestin3; A: 2S albumin; V: 7S vicilin-like protein. Numbers 1, 2, and 3 indicate DNA marker bands, representing 500, 1,000, and 2,000 bp, respectively.
Figure 5
Figure 5
Alignment results of the coding sequences.(A) Alignment result for Cs2S and soybean 2S albumin.(B) Alignment results for the three Edestins and soybean 11S globulin. Broken lines were introduced to maximize similarity. Asterisks indicate identical amino acids.(C) Alignment result of Cs7S and soybean 7S vicilin-like protein (XP_003542001). The cleavage site of signal peptide is indicated by open triangle, while the conserved cysteine residues are boxed. The black arrows indicate cleavage site for splitting these polypeptides into an acidic subunit and a basic subunit. The underlines indicate the predicted glycosylation sites.
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