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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (9): 2552-2561.doi: 10.3724/SP.J.1006.2023.21064

• 耕作栽培·生理生化 • 上一篇    下一篇

淀粉特性及其表面结合蛋白与裸燕麦籽粒硬度的关系研究

南金生1(), 安江红1,2, 柴明娜1, 蒋屿潋1, 朱志强1, 杨燕1, 韩冰1,*()   

  1. 1内蒙古农业大学 / 麦类种质创新利用自治区高等学校重点实验室, 内蒙古呼和浩特 010018
    2内蒙古自治区农牧业科学院, 内蒙古呼和浩特 010018
  • 收稿日期:2022-10-05 接受日期:2023-02-10 出版日期:2023-09-12 网络出版日期:2023-02-27
  • 通讯作者: *韩冰, E-mail: hb_nmg@163.com
  • 作者简介:南金生, E-mail: 470917658@qq.com
  • 基金资助:
    本研究由国家重点研发计划项目(2022YFE0119800);麦类种质创新利用自治区高等学校重点实验室和“饲用作物及有益微生物种质资源与分子育种”团队经费项目(TD202103)

Relationship between the starch properties and its surface-bound proteins in grains with hardness in Avena nuda L.

NAN Jin-Sheng1(), AN Jiang-Hong1,2, CHAI Ming-Na1, JIANG Yu-Lian1, ZHU Zhi-Qiang1, YANG Yan1, HAN Bing1,*()   

  1. 1Key Laboratory of Wheat Germplasm Innovation and Utilization Autonomous Region Higher School / Inner Mongolia Agricultural University, Hohhot 010018, Inner Mongolia, China
    2Inner Mongolia Academy of Agriculture and Animal Husbandry Science, Hohhot 010018, Inner Mongolia, China
  • Received:2022-10-05 Accepted:2023-02-10 Published:2023-09-12 Published online:2023-02-27
  • Supported by:
    National Key Research and Development Program of China(2022YFE0119800);Key Laboratory of Wheat Germplasm Innovation and Utilization Autonomous Region Higher School, and Forage Crops and Beneficial Microorganism Germplasm Resources and Molecular Breeding Team Funding(TD202103)

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摘要：

籽粒硬度可以反映麦类籽粒胚乳质地, 与其碾磨性能和食用加工品质密切相关。为了探究裸燕麦(Avena nuda L.)籽粒淀粉特性及其表面结合蛋白与籽粒硬度的关系。本文以5份软质和5份硬质裸燕麦种质为材料, 测定总淀粉、支链和直链淀粉含量, 观察淀粉粒特性和粒度, 对淀粉颗粒表面结合蛋白进行质谱鉴定。结果表明: 成熟籽粒中淀粉颗粒的形状有圆形、椭圆形和不规则形, 淀粉粒的数量、体积和表面积均呈现单峰分布; 淀粉粒大小可分为小(粒径<6 μm)、中(6~40 μm)和大淀粉粒(粒径>40 μm), 裸燕麦籽粒主要由小和中淀粉粒组成; 硬度不同的裸燕麦籽粒中淀粉的粒度分布不同, 软质裸燕麦小淀粉粒的数量和体积百分比高于硬质, 中、大淀粉粒的数量和体积百分比低于硬质; 软质裸燕麦小和中淀粉粒的表面积百分比高于硬质, 大淀粉粒表面积百分比低于硬质; 裸燕麦籽粒硬度与支链淀粉含量呈正相关。软质裸燕麦14 kD蛋白在淀粉颗粒表面的含量高于硬质裸燕麦, 该蛋白条带经HPLC/MS共鉴定到41种蛋白, 包括Vromindoline蛋白和燕麦α淀粉酶胰蛋白酶抑制剂等。综上所述, 裸燕麦籽粒淀粉含量、粒度分布和淀粉表面结合蛋白均与籽粒硬度相关, 支链淀粉含量越高, 小淀粉粒的数量、表面积和体积百分比越多, Vromindoline等蛋白含量越高, 裸燕麦籽粒越软, 为揭示裸燕麦籽粒硬度形成机制奠定了基础。

关键词: 裸燕麦, 籽粒硬度, 淀粉粒度, Vromindoline, HPLC/MS

Abstract:

Kernel hardness can reflect the texture of wheat kernels and it is closely related to its milling performance and edible quality. The objective of this study is to explore the relationship between naked oat (Avena nuda L.) grain starch properties and its surface-bound proteins with grain hardness. In this experiment, to determine the content of total starch, amylopectin and amylose, observe the characteristics and particle size of starch granules, and identify the surface-bound proteins of starch granules by mass spectrometry, five soft and five hard naked oat germplasms were used as the materials. The results showed that the shapes of starch granules in mature grains were round, oval and irregular, and the number, volume and surface area of starch granules showed a unimodal distribution. The size of starch granules could be divided into small (particle size < 6 μm), medium (6-40 μm), and large starch granules (particle size > 40 μm). Naked oat grains were mainly composed of small and medium starch granules. The grain size distribution of starch in naked oat grains with different hardness was different. The number and volume percentage of small starch granules in soft naked oat were higher than those in hard, and the number and volume percentage of medium and large starch grains were lower than those in hard. The percentage of surface area of soft naked oat small and medium starch granules was higher than that of hard, and the percentage of surface area of large starch granules was lower than that of hard. There was a significant positive correlation between the grain hardness of naked oat and the amylopectin content. The results showed that the protein content of the starch granule surface with a size of 14 kD was higher in soft oats than in hard ones. A total of 41 proteins were identified by HPLC/MS for starch granule surface-binding proteins at 14 kD, including Vromindoline protein and oat alpha amylase trypsin inhibitor. The above results showed that the starch content, particle size distribution and starch surface-bound protein in naked oat grains were all related to grain hardness. The higher the amylopectin content, the greater the number, surface area and volume distribution of small starch granules. The higher the protein content such as Vromindoline, and the softer the naked oat kernel. This study laid the foundation for revealing the formation mechanism of grain hardness in Avena nuda L.

Key words: Avena nuda L., grain hardness, starch particle size, Vromindoline, HPLC/MS

表1

10份裸燕麦材料信息"

组别
Group		 材料编号
Material code		 材料名称
Material name		 硬度均值
Average hardness (N)		 样品类型
Variety type		 来源
Source
软质组
Soft group		 HX317 73014-336 18.62 选育 Breeding 山西 Shanxi
HX88 燕麦 Oat 20.08 地方品种 Landrace 陕西 Shaanxi
HX106 丽江燕麦 Lijiang oat 21.62 地方品种 Landrace 云南 Yunnan
HX54 小莜麦 Little oat 21.70 地方品种 Landrace 山西 Shanxi
HX344 8399-3-4 21.89 选育 Breeding 山西 Shanxi
硬质组
Hard group		 HX310 晋8713-1 Jin 8713-1 34.69 选育 Breeding 山西 Shanxi
HX268 蒙燕8202 Mengyan 8202 35.03 地方品种 Landrace 内蒙古 Inner Mongolia
HX21 冀杂2号 Jiza 2 35.47 选育 Cultivar 河北 Hebei
HX265 蒙燕7805 Mengyan 7805 36.05 地方品种 Landrace 内蒙古 Inner Mongolia
HX320 7929-1-6 37.26 选育 Breeding 山西 Shanxi

图1

10份裸燕麦成熟籽粒中总淀粉、直链淀粉和支链淀粉的含量 不同小写字母表示在0.05概率水平差异显著。缩写同表1。"

图2

软质HX317和硬质HX320籽粒淀粉颗粒的显微结构 a~e: HX317的淀粉颗粒; f~j: HX320的淀粉颗粒; 从左向右的5列图片比例尺分别为20、10、3、1和5 μm。"

图3

HX317和HX320淀粉粒的数量、表面积和体积分布"

表2

HX317和HX320淀粉粒的数量、表面积和体积分布百分比"

淀粉粒度分布
Starch particle size distribution		 材料名称
Material name		 硬度值
Hardness value		 小淀粉粒
Small starch granules
( < 6 μm)		 中淀粉粒
Medium starch granules (6-40 μm)		 大淀粉粒
Large starch granules
( > 40 μm)
数量
Number		 HX317 18.62 N 62.86 37.14 0.006
HX320 37.26 N 48.98 50.98 0.038
表面积
Surface area		 HX317 18.62 N 0.027 99.79 0.016
HX320 37.26 N 0.009 98.26 1.730
体积
Volume		 HX317 18.62 N 14.37 84.82 0.80
HX320 37.26 N 7.59 88.89 3.52

图4

不同硬度裸燕麦中TritonX-114可溶性蛋白的SDS-PAGE a和b为全粉中和淀粉颗粒表面结合的蛋白; M为80 kD的蛋白marker; 1~10分别代表HX317、HX88、HX106、HX54、HX344、HX310、HX268、HX21、HX265和HX320, 越向右硬度值越大。"

表3

14 kD处蛋白的部分质谱分析结果"

蛋白编号
Protein number		 蛋白名称
Protein name		 分子量
Molecular weight		 蛋白打分值
Protein score		 序列覆盖率
Sequence coverage		 来源
Source
I2E102 Vromindoline 17.0 1027 48 A. barbata
U5ZZB2 Vromindoline 2 17.0 738 48 A. insularis
I2E103 Vromindoline 14.1 604 57 A. eriantha
R4I506 Vromindoline 1.3 16.8 602 33 A. sativa
I2E0Z8 Vromindoline 16.8 527 29 A. strigosa
R4I3J6 Vromindoline 3.1 15.9 527 48 A. sativa
R4I3J4 Vromindoline 1.2 16.8 462 29 A. sativa
I2E108 Vromindoline 15.8 406 48 A. marcoccana
R4I508 Vromindoline 3.2 15.9 333 46 A. sativa
A0A1B2LQE3 Avena alpha amylase trypsin inhibitor 17.2 351 41 A. insularis
A0A1B2LQE9 Avena alpha amylase trypsin inhibitor 17.1 321 41 A. insularis
A0A1B2LQE6 Avena alpha amylase trypsin inhibitor 16.9 179 35 A. magna
A0A1B2LQB5 Avena alpha amylase trypsin inhibitor 16.1 161 33 A. marcoccana
A0A1B2LQF0 Avena alpha amylase trypsin inhibitor 17.1 146 32 A. barbata
A0A1B2LQD6 Avena alpha amylase trypsin inhibitor-2 16.5 101 30 A. sativa
A0A1B2LQB9 Avena alpha amylase trypsin inhibitor 15.9 99 27 A. murphyi
A0A1B2LQD2 Avena alpha amylase trypsin inhibitor-2 16.3 89 30 A. barbata
I4EP66 Avenin 28.6 318 24 A. insularis
I4EP56 Avenin 29.2 300 22 A. longiglumis
G8ZCT8 Avenin (fragment) 28.2 240 14 A. canariensis
G8ZCU6 Avenin (fragment) 24.3 213 19 A. prostrata
L0L4J7 Gliadin-like 24.5 262 19 A. sativa
L0L833 Gliadin-like 21.4 65 8 A. sativa
O49258 12S globulin 58.2 579 20 A. sativa
O49257 12S globulin 53.4 562 26 A. sativa
P14812 12S seed storage globulin 2 58.6 749 31 A. sativa
Q38780 11S globulin 59.4 604 23 A. sativa

图5

I2E102和A0A1B2LQE3特异肽段的质谱图"

图6

9个Vromindoline蛋白和8个AATI蛋白的序列比对 A: 9个VIN蛋白和8个AATI蛋白的进化树; B: VIN蛋白的序列比对, 红框为色氨酸丰富区。"

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