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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (7): 966-976.doi: 10.3724/SP.J.1006.2018.00966

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Efficient Separation and Identification of High Molecular Weight Glutenin Subunits by HPCE

Wei-Dong WANG,Xiang GAO(),Dan-Yang ZHAO   

  1. College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, China
  • Received:2017-11-24 Accepted:2018-03-26 Online:2018-07-10 Published:2018-07-19
  • Contact: Xiang GAO E-mail:gx@nwsuaf.edu.cn
  • Supported by:
    This study was supported by the China Agriculture Research System (CARS-3-2-47) and the National High Technology Research and Development Program of China (2011AA100501).

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Abstract:

High molecular weight glutenin subunit (HMW-GS) is important for processing quality of wheat. High performance capillary electrophoresis (HPCE) is increasingly used in the work of separation and identification due to its advantages of small sample, rapidness and high precision. However, this technique has been seldom used in wheat HMW-GS study and its separation system needs improvement in analytical speed and discernibility. On the basis of HMW-GS identification with SDS-PAGE and molecular markers, an high-efficiency separation system of HPCE was set up using Chinese Spring as the standard. The system components (pH 2.5) were 75 mmol L -1 IDA, 0.05% HPMC, and 15% ACN. The electrophoresis parameters were 25 μm of inner diameter of the capillary, 200 nm of detection wavelength, 20 kV of separation voltage, and 30°C of operating temperature. Using mixed injection method, the standard spectrums were obtained for 18 subunits. Their migration order was 1Dy12→ 1Dy10→ 1By9→ 1By8→ 1By18→ 1By16→ 1By20→ 1Bx17→ 1Bx20→ 1Bx13→ 1Bx6→ 1Bx7→ 1Ax2*→ 1Ax1→ 1Dx5→ 1Dx4→ 1Dx3→ 1Dx2, and standard peak time of these subunits was 9.39, 9.69, 10.30, 11.70, 11.89, 12.09, 12.22, 12.36, 12.62, 12.83, 13.08, 13.18, 13.50, 13.73, 14.04, 14.24, 14.46, and 14.73 min, respectively. The relative standard deviation was smaller than 0.2%. The y-type and x-type subunits appeared in the phases of 9.39-12.36 min and 12.36-14.76 min, respectively, between which there was 1Bx17 as the boundary. These results indicate that the HPCE separation system is applicable in rapid identification of HMW-GS in wheat germplasm resources when we simultaneously consider migration order, standard peak time, and HPCE spectrum.

Key words: wheat, high molecular weight glutenin subunit, HPCE spectrum, high efficiency separation system of HPCE

Table 1

Molecular markers of HMW-GS"

亚基
Subunit		 标记序列
Sequence (5′→3′)		 片段大小
Size of
fragment (bp)		 参考文献
Reference
1Ax1 F: TCACCGACAGTCCACCGA; R: ACCAAGCGAGCTGCAGAG 2532 Bustos et al. [16]
Null F: ACGTTCCCCTACAGGTACTA; R: TATCACTGGCTAGCCGACAA 920 Lafiandra et al. [17]
1Ax2* F: ATGACTAAGCGGTTGGTTCTT; R: ACCTTGCTCCCCTTGTCTTT 1319 Ma et al. [18]
1Bx6 F: CACTGAGATGGCTAAGCGCC; R: GCCTTGGACGGCACCACAGG 321 Schwarz et al. [19]
1Bx7/1Bx17 F: CGCAACAGCGAGGACAATT; R: TGGTCCGTCACTATCTTGAGA 766/669 Ma et al. [18]
1Bx13
 F: ATGAGCTAAGCGCGCTGGTCCTCTTTG;
R: CTATCACTGCCTGGTCCGACAATGCG		 900
 Pang and Zhang [20]
1Bx20 F: CCTCAGCATGCAAACATGCAGC; R: CTGAAACCTTTGGCCAGTCATGTC 800 Butow et al. [21]
1By8 F: TTAGCGCTAAGTGCCGTCT; R: TTGTCCTATTTGCTGCCCTT 527 Lei et al. [22]
1By9 F: TTCTCTGCATCAGTCAGGA; R: AGAGAAGCTGTGTAATGCC 707 Lei et al. [22]
1By16 F: GCAGTACCCAGCTTCTCAA; R: CCTTGTCTTGTTTGTTGCC 3 fragments Lei et al. [22]
1By18 F: CAACAAAACGGGCGTTGT; R: CAACAAAACGGGCGTTGT 365 Liang et al. [23]
1Dx2/1Dx5 F: GGGACAATACGAGCAGCAAA; R: CTTGTTCCGGTTGTTGCCA 299/281 Liu et al. [24]
1Dy10/1Dy12 F: GTTGGCCGGTCGGCTGCCATG; R: TGGAGAAGTTGGATAGTACC 576/612 Ahmad [25]

Fig. 1

Identification of the subunit composition and coding genes of Glu-1 loci in wheatA: analysis of SDS-PAGE; B-F: identification of HMW-GS by molecular markers. M: Trans 2k plus DNA marker; 1: Chinese Spring; 2: Xinong 979; 3: Jinan 13; 4: Jinmai 47; 5: Jimai 4; 6: Yumai 41; 7: Yannong 19; 8: Laizhou 137; 9: Lumai 23; 10: Aikang 58; 11: Yumai 50; 12: Zhengmai 366."

Table 2

Composition of HMW-GS of material varieties"

品种 Variety Glu-1A Glu-1B Glu-1D
中国春 Chinese Spring null 7+8 2+12
西农979 Xinong 979 1 7+8 2+12
济南13 Jinan 13 1 7+9 2+12
晋麦47 Jinmai 47 null 7+9 3+12
济麦4号 Jimai 4 1 13+16 4+12
豫麦41 Yumai 41 1 20x+20y 5+10
烟农19 Yannong 19 1 17+18 4+12
莱州137 Laizhou 137 1 6+8 5+10
鲁麦23 Lumai 23 null 20 2+12
矮抗58 Aikang 58 1 7+8 4+12
豫麦50 Yumai 50 2* 7+9 2+12
郑麦366 Zhengmai 366 1 7+8 5+10

Fig. 2

Effect of constituent concentration and pH values of buffer on electrophoresis separation of HMW-GSA: effect of different pH on the electrophoresis separation, the constituents of the buffer were 100 mmol L-1 IDA + 0.05% HPMC + 20% ACN. B: results of two successive electrophoretic separation under different concentrations of IDA, the other constituents of the buffer were 0.05% HPMC + 20% ACN, the pH was 2.5. C: effect of different concentrations of HPMC on the electrophoresis separation, the other constituents of the buffer were 75 mmol L-1 IDA + 20% ACN, the pH was 2.5. D: effect of different concentrations of ACN on the electrophoresis separation, the other constituents of the buffer were 75 mmol L-1 IDA + 0.05% HPMC, the pH was 2.5. Electrophoresis parameters of A-D: the inner diameter of the capillary was 50 μm; the detection wavelength was 214 nm; the separation voltage was 15 kV; the operating temperature was 25 °C. The arrow indicates the position of the secondary peak."

Fig. 3

HPCE profile before (A) and after (B) optimization of electrophoresis parametersBuffer constituents: 75 mmol L-1 IDA + 0.05% HPMC + 15% ACN, pH 2.5. Electrophoresis parameters were 50 μm of the inner diameter of capillary, 214 nm of detection wavelength, 15 kV of separation voltage, and 25°C of operating temperature before optimization and 25 μm of the inner diameter of capillary, 200 nm of the detection wavelength, 20 kV of separation voltage, and 30°C of operating temperature after optimization."

Fig. 4

Thirty separations of HMW-GS of Chinese Spring by HPCEThe curves correspond to the 1st, 5th, 10th, 15th, 12th, 25th, and 30th electrophoresis separation, respectively."

Fig. 5

Separation profile of HPCE (A) and RP-HPLC (B)Chinese Spring HMW-GS was the standard. Nomenclature of characteristic peaks in the RP-HPLC profile follows the descriptions of Yan et al. [34]"

Table 3

Relative standard deviation and peak time of HMW-GS"

亚基
Subunit		 HPCE RP-HPLC
出峰时间
Peak time (min)		 相对标准偏差
Relative standard deviation (%)		 出峰时间
Peak time (min)		 相对标准偏差
Relative standard deviation (%)
1Dx2 14.73±0.02 0.12 22.31±0.11 0.40
1Bx7 13.18±0.01 0.07 28.42±0.22 0.63
1By8 11.70±0.01 0.14 21.86±0.21 0.78
1Dy12 9.39±0.02 0.18 15.91±0.18 0.92

Fig. 6

HPCE spectrum of different HMW-GS (mixed injection)A: Chinese Spring (null, 7+8, 2+12); B: Chinese Spring + Xinong 979 (1, 7+8, 2+12); C: Chinese Spring + Xinong 979 + Jinan 13 (1, 7+9, 2+12); D: Chinese Spring + Xinong 979 + Jinan 13; E: Chinese Spring + Xinong 979 + Jinan 13 + Yumai 50 (2*, 7+9, 2+12); F: Chinese Spring + Xinong 979 + Jinan 13 + Jinmai 47 (null, 7+9, 3+12); G: Chinese Spring; H: Chinese Spring + Zhengmai 366 (1, 7+8, 5+10); I: Chinese Spring + Zhengmai 366 + Yumai 41 (1, 20x+20y, 5+10); J: Chinese Spring + Zhengmai 366; K: Chinese Spring + Zhengmai 366 + Laizhou 137 (1, 6+8, 5+10); L: Chinese Spring + Zhengmai 366+Laizhou 137+Lumai 23 (Null, 20, 2+12). M: Chinese Spring + Xinong 979; N: Chinese Spring + Xinong 979+Aikang 58 (1, 7+8, 4+12); O: Chinese Spring + Xinong 979+Aikang 58+Jimai 4 (1, 13+16, 4+12); P: Chinese Spring + Xinong 979 + Aikang 58 + Yannong 19 (1, 17+18, 4+12)."

Table 4

Peak time of eighteen HMW-GSs"

Glu-1位点
Glu-1 locus		 亚基类型
Type of subunit		 亚基
Subunit		 出峰时间
Peak time (min)
Glu-1A x-type 1Ax1 13.73
1Ax2* 13.50
Glu-1B x-type 1Bx6 13.08
1Bx7 13.18
1Bx13 12.83
1Bx20 12.62
1Bx17 12.36
y-type 1By8 11.70
1By9 10.30
1By20 12.22
1By16 12.09
1By18 11.89
Glu-1D x-type 1Dx2 14.73
1Dx3 14.46
1Dx4 14.24
1Dx5 14.04
y-type 1Dy10 9.69
1Dy12 9.39
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