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作物学报 ›› 2021, Vol. 47 ›› Issue (7): 1248-1258.doi: 10.3724/SP.J.1006.2021.01062

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

不同耐旱性青稞叶片差异蛋白分析

李洁, 付惠, 姚晓华, 吴昆仑*()   

  1. 青海大学农林科学院 / 青海省农林科学院 / 青海省青稞遗传育种重点实验室 / 国家麦类改良中心青海青稞分中心, 青海西宁810016
  • 收稿日期:2020-08-05 接受日期:2020-12-01 出版日期:2021-07-12 网络出版日期:2021-03-14
  • 通讯作者: 吴昆仑
  • 作者简介:李洁, E-mail: lij_28257@163.com
  • 基金资助:
    本研究由国家自然科学基金项目(32060480);本研究由国家自然科学基金项目(31660388);本研究由国家自然科学基金项目(31960427);青海省农林科学院创新基金(2018-NKY-12);国家现代农业产业技术体系建设专项(CARS-05);青海省科技厅应用基础研究项目资助(2019-ZJ-7075)

Differentially expressed protein analysis of different drought tolerance hulless barley leaves

LI Jie, FU Hui, YAO Xiao-Hua, WU Kun-Lun*()   

  1. Academy of Agricultural and Forestry Sciences, Qinghai University / Qinghai Academy of Agricultural and Forestry Sciences / Qinghai Key Laboratory of Hulless Barley Genetics and Breeding / Qinghai Subcenter of National Hulless Barley Improvement, Xining 810016, Qinghai, China
  • Received:2020-08-05 Accepted:2020-12-01 Published:2021-07-12 Published online:2021-03-14
  • Contact: WU Kun-Lun
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(32060480);This study was supported by the National Natural Science Foundation of China(31660388);This study was supported by the National Natural Science Foundation of China(31960427);the Qinghai Provincial Academy of Agriculture and Forestry Innovation Fund(2018-NKY-12);the China Agriculture Research System(CARS-05);the Qinghai Science and Technology Support Project(2019-ZJ-7075)

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摘要：

为从蛋白质水平揭示不同青稞品种响应干旱胁迫的差异, 分析抗旱蛋白质分子机制, 本研究以干旱胁迫不敏感的旱地紫青稞(HDZ)和干旱胁迫敏感的大麻青稞(DM)为研究材料, 以盆栽限量供水种植方法, 对干旱处理不同梯度的青稞叶片进行叶绿素、可溶性蛋白、丙二醛含量及相对电导率4项生理指标的测定, 同时利用iTRAQ技术对深度干旱胁迫青稞叶片全蛋白组进行差异蛋白分析。结果表明: 随着干旱处理时间的延长, 两种青稞的叶绿素和可溶性蛋白含量逐渐下降, 电导率及丙二醛的含量逐渐升高, 在相同处理条件下, 大麻青稞的叶绿素和可溶性蛋白含量降低幅度、电导率及丙二醛含量的增高幅度明显高于旱地紫青稞; 对两个青稞品种的干旱胁迫和正常培养的比较组进行iTRAQ分析, 共定量出4163个蛋白(多肽), 其中旱地紫青稞比较组中对比正常培养, 筛选到表达上调的蛋白68个, 下调的蛋白63个, 在大麻青稞的比较组中筛选出表达上调蛋白21个, 下调蛋白32个。KEGG通路分析表明, 富集程度位于前3位的通路是代谢、氨基酸的生物合成以及次级代谢产物的生物合成, 主要涉及到柠檬酸循环、碳循环等代谢通路, 丙氨酸、精氨酸等氨基酸的合成降解以及花生四烯酸、亚麻酸等次级代谢产物的合成。本研究从蛋白组水平筛选了青稞干旱胁迫响应相关的代谢通路和其他相关功能的蛋白, 为揭示青稞干旱胁迫的应答分子调控机制提供了一定的理论基础。

关键词: 青稞, 干旱胁迫, 叶片, 差异蛋白, iTRAQ

Abstract:

In order to reveal the differences in response to drought stress among different hulless barley varieties from the protein level and to analyze the protein molecular mechanism of drought tolerance, Handizi Barley (HDZ) resistant to drought stress and Dama Barley (DM) sensitive to drought stress were used as research materials in this study. Drought treatment were determined by potted-planting method with limited water supply, four physiological indexes of hulless barley leaves with different drought gradients, including chlorophyll, soluble protein, malondialdehyde content, and relative conductivity were investigated. iTRAQ technology was used to conduct differential protein analysis on the whole protein group of barley leaves under deep drought stress. The results showed that with the extension of the drought treatment, the chlorophyll and soluble protein content of two hulless barleys under drought stress gradually decreased, the electrical conductivity and malondialdehyde content gradually increased, and the decrease in chlorophyll and soluble protein content, the increase in electrical conductivity and the content of malondialdehyde in Dama were greater than that of Handizi; 4163 proteins (polypeptides) were quantified, among them, compared with normal culture in the Handizi comparison group, 68 up-regulated proteins and 63 down-regulated proteins were screened by iTRAQ; in the comparative group of Dama, 21 up-regulated proteins and 32 down-regulated proteins were screened. KEGG pathway showed that the top three enrichment pathways were metabolic, amino acid biosynthesis, and secondary metabolite biosynthesis. The first one mainly related to citric acid cycle, carbon cycle, and other metabolic pathways. The synthesis and degradation of amino acids were mainly involved arginine and alanine. The synthesis of secondary metabolites were about arachidonic acid and linolenic acid. This study screened the proteins related to the metabolic pathways and other related functions in response to drought stress on proteome level in hulless barley, providing a theoretical basis for revealing the molecular regulation mechanism in response to drought stress.

Key words: hulless barley, drought stress, leaf, differentially expressed protein, iTRAQ

图1

HDZD-HDZCK和DMD-DMCK叶绿素含量变化 A: 叶绿素a; B: 叶绿素b; C: 总叶绿素。大写字母表示0.01极显著水平, 小写字母表示0.05显著水平。"

表1

干旱处理对青稞叶片蛋白质、相对电导率及丙二醛含量的影响"

处理
Treatment		 品种
Variety		 蛋白质含量
Protein content
(mg g-1)		 相对电导率
Relative electrical
conductivity (%)		 丙二醛含量
Malondialdehyde content
(μmoL g-1)
对照
CK		 旱地紫 HDZ 7.30±0.10 Aa 44.50±0.68 Cc 32.92±0.45 Cc
大麻 DM 10.00±0.56 Aa 50.10±0.34 Cc 28.70±0.10 Cc
干旱处理7 d
7 days after drought treatment		 旱地紫 HDZ 6.90±0.55 Aa 45.89±0.47 Bb 35.55±0.29 Bb
大麻 DM 7.87±0.12 Bb 54.33±0.60 Bb 32.21±0.38 Bb
干旱处理10 d
10 days after drought treatment		 旱地紫 HDZ 6.75±0.26 Aa 48.97±0.71 Aa 38.92±0.42 Aa
大麻 DM 7.65±0.61 Bb 60.22±0.39 Aa 40.33±0.48 Aa

表2

鉴定蛋白的覆盖率分布"

比较组
Comparison group		 肽段覆盖率 Protein coverage (%) 总计
Total
≤10 10<X≤20 20<X≤30 30<X≤40 40<X≤50 50<X≤60 60<X≤70
HDZD-HDZCK 650 807 409 192 64 18 8 2150
DMD-DMCK 622 752 390 170 55 16 6 2013

图2

HDZD-HDZCK青稞(左)和DMD-DMCK青稞(右)差异表达蛋白火山图"

图3

GO富集柱状图 A: 属于生物过程(BP); B: 属于细胞组分(CC); C、D、E、F、G属于分子功能(MF); A: 防御响应; B: 胞外部分; C: 催化活性; D: ATP结合; E: 结构分子活性; F: 转移活性; G: 运输活性; H: 其他活性。"

表3

HDZD-HDZCKA和DMD-DMCK差异表达蛋白功能分类"

蛋白质功能分类
Functional classification of proteins		 蛋白质数量
Number of proteins
胁迫应答类 Stress response 11
脂类代谢相关类 Lipid metabolism 13
糖类代谢相关类 Carbohydrate metabolism 10
蛋白质代谢相关类 Protein metabolism 48
氧化还原类 Oxidoreduction 35
能量供应类 Energy 16
光合相关类 Photosynthetic 15
未知蛋白 Unknown functional protein 36

图4

HDZD-HDZCK(左)和DMD-DMCK(右)差异蛋白KEGG注释"

表4

显著差异蛋白的KEGG通路"

通路ID
Pathway ID		 通路名称
Pathway name
ko00020 三羧酸循环 TCA cycle
ko00640 丙酸酯代谢 Propanoate metabolism
ko01200 碳代谢 Carbon metabolism
ko00270 半胱氨酸和蛋氨酸代谢 Cysteine and methionine metabolism
ko00620 丙酮酸代谢 Pyruvate metabolism
ko00630 乙醛酸和二羧酸的代谢 Glyoxylate and dicarboxylate metabolism
ko00710 光合作用中的碳固定 Carbon fixation in photosynthetic
ko00195 光合作用 Photosynthsis
ko03010 核糖体 Ribosome
ko03013 RNA转运 RNA transport
ko00190 氧化磷酸化 Oxidaxtive phosphorylation
ko04145 吞噬作用 Phagosome
ko04141 内质网的蛋白质加工 Protein processing in endoplasmic reticulum
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