Increased sulfur-containing amino acid content and altered conformational characteristics of soybean proteins by rebalancing 11S and 7S compositions

Abstract

Soybean proteins are limited by their low contents of methionine and cysteine. Herein, 7S globulin accumulation was reduced using RNA interference to silence CG-β-1 expression, and the content of the A2B1a subunit was largely increased under the soybean seed-specific oleosin8 promoter. The results showed that the sulfur-containing amino acid content in soybean seeds drastically improved, reaching 79.194 nmol/mg, and the 11S/7S ratio had a 1.89-fold increase compared to the wild-type acceptor. The secondary structures of 11S globulin were also altered, and the β-sheet content increased with decreasing β-turn content, which was confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy and circular dichroism analysis. Our findings suggested that raising the accumulation of 11S glycinin at the expense of reducing the content of 7S globulin is an attractive and precise engineering strategy to increase the amount of sulfur-containing amino acids, and soybean proteins with A2B1a subunits of 11S isolates improved, and β-subunits of 7S fractions reduced simultaneously might be an effective new material for food production.

Keywords: 11S glycinin; 7S globulin; RNA interference; conformational characteristics; soybean proteins; sulfur-containing amino acids.

Figure 1

Schematic diagram of pCambia1300-OsEPSPS-RNAi-GmOle8pro-A2B1a. The soybean transformation construct contains the A2B1a subunit coding region (A2B1a ORF) under the control of the soybean oleosin8 promoter (GmOle8pro) and the 3′-end noncoding region of 2S albumin (2S terminator). The RNAi cassette under the control of the soybean α-promoter of β-conglycinin and the octopine synthase terminator, together with 302 bp fragments obtained from sequences of CG-β-1 gene forward and reverse inserted side of PDK intron, respectively. The OsEPSPS gene under the control of the cauliflower mosaic virus 35S promoter (CaMV35S) and 35S poly(A) terminator acted as a selective marker. EcoRI, KpnI, BamHI and PstI restriction sites are shown on the vector. LB, left border; RB, right border.

Figure 2

Expression of OsEPSPS, CG-β-1 and gy2 in transgenic soybean plants. (A) Detection of OsEPSPS transcripts by PCR amplification. M, DNA marker 5,000; N, the negative control ‘Tianlong No. 1’; P, the positive plasmid; Lane 1, T1000 transgenic lines; Lane 2, T1006 transgenic lines. (B) Glyphosate screening for transgenic lines. CK, ‘Tianlong No. 1’ without spraying glyphosate; T1000, T1000 transgenic lines sprayed with glyphosate (10 ml/l); T1006, T1006 transgenic lines sprayed with glyphosate (10 ml/l). (C) Detection of CG-β-1 and gy2 expression using quantitative real-time PCR. CK, T1000, and T1006 represent ‘Tianlong No. 1’, T1000, and T1006 transgenic plants, respectively, and the levels of CG-β-1 and gy2 measured in ‘Tianlong No. 1’ were regarded as the control.

Figure 3

Analysis of the subunit composition of 7S β-conglycinin and 11S glycinin in soybean seeds. (A,B) are standard calibration curves of competitive ELISA for 7S and 11S globulins using polyclonal antibodies, respectively. (C) The content of 7S β-conglycinin and 11S glycinin in dry soybean seeds from different plants determined by ELISA results (3 replicates). The letters indicate the level of significance at the level p = 0.01. (D) Typical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE, 12%) profiles of total proteins obtained from dry soybean seeds. The bands of the β subunit and the acidic and basic components of 11S globulins were noted. Lane 1, Lane 2, Lane 3, and Lane 4 represent T1000 and T1006 transgenic plants, ‘Tianlong No. 1’ and protein markers, respectively.

Figure 4

Gel permeation chromatography of SPI obtained from different plants. (A–C) are molecular weight (Mw) distribution profiles for the ‘Tianlong No. 1’, T1000, T1006 lines, respectively. (D) The proteins collected from elution times of 5.0–6.5 min were separated by SDS–PAGE. Lane 1, Lane 2, Lane 3, and Lane 4 represent the protein marker, T1000 lines, ‘Tianlong No. 1’, and T1006 transgenic plants, respectively. (E) The area distribution of 7S, 11S acidic, and 11S basic fractions from ‘Tianlong No. 1’, T1006 and T1000 plants, respectively.

Figure 5

Circular dichroism, FTIR and Raman spectra of 7S and 11S isolates from different lines. (A,B) CD spectra; (C,D) FTIR spectra; (E,F) Raman spectra; CK, T1000, T1006 represent the ‘Tianlong No. 1’, T1000, T1006 lines, respectively. (G) Disulfide bond configuration of 7S and 11S fractions from different samples. Different letters indicate the level of significance at the level p = 0.05. (H) Secondary structure content of 7S and 11S fractions from different samples determined by CD, FTIR and Raman spectra. Different letters (a–c) indicate significant differences (p = 0.05).