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作物学报 ›› 2018, Vol. 44 ›› Issue (7): 966-976.doi: 10.3724/SP.J.1006.2018.00966

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

高分子量麦谷蛋白亚基HPCE高效分离及图谱鉴定

王卫东,高翔(),赵丹阳   

  1. 西北农林科技大学农学院, 陕西杨凌 712100
  • 收稿日期:2017-11-24 接受日期:2018-03-26 出版日期:2018-07-10 网络出版日期:2018-07-19
  • 通讯作者: 高翔
  • 基金资助:
    本研究由国家现代农业产业技术体系建设专项(CARS-3-2-47)和国家高技术研究发展计划(863计划)项目(2011AA100501)资助

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 Published:2018-07-10 Published online:2018-07-19
  • Contact: Xiang GAO
  • 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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摘要：

高分子量麦谷蛋白亚基(HMW-GS)是决定小麦加工品质的重要因素。高效毛细管电泳技术(HPCE)以其用样少、速度快、精确度高等特点被越来越多地应用到分离鉴定中来。目前小麦HMW-GS的HPCE研究尚处于起步阶段, 对标准鉴定图谱的研究甚少,且已有分离体系在连续多次试验中的分离速度及分辨率仍有提升空间。本研究通过SDS-PAGE结合分子标记的方法鉴定了不同小麦品种的HMW-GS, 以“中国春”小麦为标样确立了HPCE高效分离体系, 体系条件为75 mmol L-1 IDA+0.05% HPMC+15% ACN, pH 2.5, 电泳参数为毛细管内径25 μm, PDA检测波长200 nm, 分离电压20 kV, 运行温度30°C。通过混合进样方式对不同类型亚基进行HPCE分析, 建立了18个亚基的标准图谱, 亚基迁移顺序为1Dy12→ 1Dy10→ 1By9→ 1By8→ 1By18→ 1By16→ 1By20→ 1Bx17→ 1Bx20→ 1Bx13→ 1Bx6→ 1Bx7→ 1Ax2*→ 1Ax1→ 1Dx5→ 1Dx4→ 1Dx3→ 1Dx2,标准出峰时间依次为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和14.73 min, RSD<0.2%。以1Bx17为分界线, 9.39 min到12.36 min为y型亚基区域, 12.36 min到14.76 min为x型亚基区域。结合亚基迁移顺序、标准出峰时间及图谱特点可对小麦相关HMW-GS进行HPCE快速鉴定。本研究获得的分离体系及鉴定图谱可用于小麦HMW-GS定性分析和种质资源筛选。

关键词: 小麦, HMW-GS, HPCE图谱, HPCE高效分离体系

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

表1

HMW-GS分子标记"

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]

图1

小麦Glu-1位点亚基组成和编码基因的鉴定A: SDS-PAGE分析; B~F: HMW-GS分子标记鉴定。M: Trans 2k plus; 1: 中国春; 2: 西农979; 3: 济南13; 4: 晋麦47; 5: 济麦4号; 6: 豫麦41; 7: 烟农19; 8: 州137; 9: 鲁麦23; 10: 矮抗58; 11: 豫麦50; 12: 郑麦366。"

表2

材料品种HMW-GS组成"

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

图2

缓冲液组分浓度、pH对HMW-GS电泳分离的影响A: 不同pH对分离效果的影响, 缓冲液组分为100 mmol L-1 IDA+0.05% HPMC+20% ACN。B: 缓冲液不同IDA浓度下连续两针电泳分离结果, 其他组分为0.05% HPMC+20% ACN, pH 2.5。C: 缓冲液不同HPMC浓度对分离效果的影响, 其他组分为75 mmol L-1 IDA+20% ACN, pH 2.5。D: 缓冲液不同ACN浓度对分离效果的影响, 其他组分为75 mmol L-1 IDA+0.05% HPMC, pH 2.5。A~D电泳参数均为毛细管内径50 μm, PDA检测波长214 nm, 分离电压15 kV, 运行温度25 °C。箭头所示为副峰。"

图3

电泳参数优化前(A)和优化后(B)的HPCE图谱缓冲液成分: 75 mmol L-1 IDA + 0.05% HPMC + 15% ACN, pH 2.5。优化前电泳参数: 毛细管内径50 μm, PDA检测波长214 nm, 分离电压15 kV, 运行温度25°C; 优化后电泳参数: 毛细管内径25 μm, PDA检测波长200 nm, 分离电压20 kV, 运行温度30°C。"

图4

中国春HMW-GS连续30次HPCE分离图中曲线分别对应第1、5、10、15、20、25和30次电泳分离。"

图5

HPCE (A)与RP-HPLC (B)的分离图谱以中国春HMW-GS为标准样品。参照Yan等[34]的方法命名RP-HPLC中的特征峰。"

表3

HMW-GS出峰时间相对标准偏差"

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

图6

不同HMW-GS的HPCE图谱(混合进样)A: 中国春(null, 7+8, 2+12); B: 中国春+西农979 (1, 7+8, 2+12); C: 中国春+西农979+济南13 (1, 7+9, 2+12); D: 中国春+西农979+济南13; E: 中国春+西农979+济南13+豫麦50 (2*, 7+9, 2+12); F: 中国春+西农979+济南13+晋麦47 (null, 7+9, 3+12); G: 中国春; H: 中国春+郑麦366 (1, 7+8, 5+10); I: 中国春+郑麦366+豫麦41 (1, 20x+20y, 5+10); J: 中国春+郑麦366; K: 中国春+郑麦366+莱州137 (1, 6+8, 5+10); L: 中国春+郑麦366+莱州137+鲁麦23 (null, 20, 2+12)。M: 中国春+西农979; N: 中国春+西农979+矮抗58 (1, 7+8, 4+12); O: 中国春+西农979+矮抗58+济麦4号 (1, 13+16, 4+12); P: 中国春(null, 7+8, 2+12) +西农979+矮抗58+烟农19 (1, 17+18, 4+12)。"

表4

18个HMW-GS的出峰时间"

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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