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作物学报

作物学报 ›› 2025, Vol. 51 ›› Issue (4): 1050-1060.doi: 10.3724/SP.J.1006.2025.44136

• 耕作栽培·生理生化 • 上一篇    下一篇

4种马铃薯内源激素提取方法优化及其在块茎解除休眠过程中的含量分析

赵喜娟1(), 张帆2, 刘圣宣2, 覃骏2, 陈惠兰2, 林原1, 罗红兵1, 刘易3, 宋波涛2, 胡新喜1,*(), 王恩爽2,*()   

  1. 1湖南农业大学 / 湖南省蔬菜生物学重点实验室 / 岳麓山实验室, 湖南长沙 410128
    2华中农业大学园艺林学学院 / 农业农村部马铃薯生物学与生物技术重点实验室 / 果蔬园艺作物种质创新与利用全国重点实验室, 湖北武汉 430070
    3新疆农业科学院综合试验场, 新疆乌鲁木齐 830013
  • 收稿日期:2024-08-25 接受日期:2024-12-12 出版日期:2025-04-12 网络出版日期:2025-01-02
  • 通讯作者: 胡新喜, E-mail: hu_xinxi@126.com; 王恩爽, E-mail: wangenshuang@126.com
  • 作者简介:赵喜娟, E-mail: 704986451@qq.com第一联系人：

    **同等贡献

  • 基金资助:
    新疆维吾尔自治区智力援疆创新拓展人才计划“小组团”援疆团队—“马铃薯生物技术与早熟育种团队”项目和国家重点研发计划项目(2023YFD1201505);新疆维吾尔自治区智力援疆创新拓展人才计划“小组团”援疆团队—“马铃薯生物技术与早熟育种团队”项目和国家重点研发计划项目(2022YFD1100201)

Optimization of extraction methods for four endogenous hormones in potatoes and analysis of their content during the process of releasing dormancy in tubers

ZHAO Xi-Juan1(), ZHANG Fan2, LIU Sheng-Xuan2, QIN Jun2, CHEN Hui-Lan2, LIN Yuan1, LUO Hong-Bing1, LIU Yi3, SONG Bo-Tao2, HU Xin-Xi1,*(), WANG En-Shuang2,*()   

  1. 1Hunan Agricultural University / Key Laboratory for Vegetable Biology of Hunan Province / Yuelu Mountain Laboratory, Changsha 410128, Hunan, China
    2College of Horticulture & Forestry Sciences, Huazhong Agricultural University / Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / National Key Laboratory of Germplasm Innovation and Utilization of Horticulture Crops, Wuhan 430070, Hubei, China
    3Comprehensive Test Site of Xinjiang Academy of Agricultural Sciences, Urumqi 830013, Xinjiang, China
  • Received:2024-08-25 Accepted:2024-12-12 Published:2025-04-12 Published online:2025-01-02
  • Contact: E-mail: hu_xinxi@126.com; E-mail: wangenshuang@126.com
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    Xinjiang Uygur Autonomous Region Intelligence Assistance Xinjiang Innovation and Expansion Talent Plan “Group Team” Assistance Xinjiang Team—“Potato Biotechnology and Early Maturing Breeding Team” Project and the National Key Research and Development Program of China(2023YFD1201505);Xinjiang Uygur Autonomous Region Intelligence Assistance Xinjiang Innovation and Expansion Talent Plan “Group Team” Assistance Xinjiang Team—“Potato Biotechnology and Early Maturing Breeding Team” Project and the National Key Research and Development Program of China(2022YFD1100201)

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摘要：

植物激素是生长发育的重要调节物质, 但马铃薯内源激素还没有统一、高效的检测方法, 难以满足深入研究的需求。本研究利用超高效液相色谱技术, 设计单因素试验和正交试验, 优化了SA、IAA、ABA和JA这4种植物激素的提取方法, 其重复性和稳定性的相对标准偏差均小于10%, 提取效率显著高于其他提取方法。利用该方法, 对生长30 d的马铃薯植株的根、茎、叶、匍匐茎和顶芽的激素含量进行测定发现, 顶芽和叶片中SA含量最高, 顶芽和匍匐茎中IAA含量最高, 叶片中ABA含量最高, 根和匍匐茎中JA含量偏高。对马铃薯栽培种华薯1号和中薯5号的块茎分别在4℃、22℃和28℃条件下储存, 并检测了其休眠打破过程中激素含量。 结果表明,在整个休眠期, IAA含量整体呈上升趋势, SA含量呈先上升后下降而后快速上升的趋势, ABA含量整体呈下降趋势。本研究优化的提取方法提取效率高、灵敏度高、稳定性和重复性好, 适用于马铃薯4种内源激素的检测分析, 研究数据为马铃薯不同组织和块茎休眠过程中激素合成及作用机制提供理论参考。

关键词: 马铃薯, 激素, 提取方法, 超高效液相色谱, 休眠

Abstract:

Plant hormones are essential regulators of growth and development. However, the lack of a unified and efficient method for detecting endogenous hormones in potato has hindered in-depth research. In this study, ultra-high performance liquid chromatography (UPLC) was combined with single-factor and orthogonal experimental designs to optimize the extraction methods for four phytohormones: salicylic acid (SA), indole-3-acetic acid (IAA), abscisic acid (ABA), and jasmonic acid (JA). The optimized method demonstrated excellent repeatability and stability, with relative standard deviations (RSDs) below 10%, and significantly improved extraction efficiency compared to conventional methods. Using this optimized method, the hormone contents in the roots, stems, leaves, apical buds, and stolons of potato plants at 30 days of growth were quantified. The results revealed that endogenous SA levels were highest in the apical buds and leaves, IAA was most abundant in the shoot apex and stolons, ABA was concentrated in the leaves, and JA levels were higher in the roots and stolons. Additionally, the tubers of potato cultivars Huashu 1 and Zhongshu 5 were stored at 4°C, 22℃, and 28℃, and hormone levels during dormancy breaking were analyzed. The findings showed a general upward trend in IAA levels throughout storage, a fluctuating pattern in SA levels (initial increase, subsequent decline, and rapid rise), and a consistent decrease in ABA levels. The optimized extraction method developed in this study offers high efficiency, sensitivity, stability, and repeatability, making it suitable for the detection and analysis of these four endogenous hormones in potato. This research provides valuable insights into hormone synthesis and their regulatory mechanisms during dormancy and across various tissues in potato.

Key words: potato, hormones, extraction method, ultra high-performance liquid chromatography, dormancy

表1

色谱洗脱梯度表"

序号
No.		 时间
Time (min)		 流动相A
Mobile phase A (%)		 流动相B
Mobile phase B (%)
1 0 Run
2 0 90 10
3 2.0 90 10
4 8.0 10 90
5 11.0 10 90
6 11.5 90 10
7 14.0 Stop

图1

4种激素的标准曲线 SA: 水杨酸; IAA: 吲哚-3-乙酸; ABA: 脱落酸; JA: 茉莉酸。"

表2

正交设计表"

处理编号
Treatment number		 A有机相比例
Organic phase ratio (%)		 B超声时间
Ultrasound time (min)		 C酸度
Acidification degree (%)		 D提取时间
Extraction time (h)
1 (A1B1C1D1) 80 20 0.7 9
2 (A1B2C2D2) 80 25 1.0 12
3 (A1B3C2D2) 80 30 1.0 12
4 (A2B1C2D2) 90 20 1.0 12
5 (A2B2C2D1) 90 25 1.0 9
6 (A2B3C1D2) 90 30 0.7 12
7 (A3B1C2D2) 100 20 1.0 12
8 (A3B2C1D2) 100 25 0.7 12
9 (A3B3C2D1) 100 30 1.0 9

表3

6种激素提取方法"

提取方法
Extraction method		 有机相
Organic phase		 超声时间
Ultrasound time (min)		 提取时间
Extraction time
(h)		 酸化程度
Acidification degree
1 80%甲醇水 80% Methanol water 12
2 80%异丙醇水 80% Isopropanol water 12
3 50%乙腈水 50% Acetonitrile water 12
4 100%乙腈 100% Acetonitrile 12
5 100%乙腈 100% Acetonitrile 1
6 100%乙腈 100% Acetonitrile 25 12 0.7%乙酸 0.7% Acetic acid

图2

单因素条件下E3叶片植物激素回收效果 缩写同图1。A~D依次是不同有机相比例、超声时间、酸化程度、提取时间处理的E3叶片中4种激素回收浓度。数据为3个重复的平均值±标准偏差。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.05)。"

图3

4种激素提取的正交试验效果 缩写同图1。A~D依次是不同处理下E3叶片中ABA、IAA、JA、SA的含量。横坐标中A1、A2、A3依次是有机相比例80%、90%、100%; B1、B2、B3依次是超声20、25和30 min; C1、C2依次是酸化程度0.7%、1.0%; D1、D2依次是提取时间9 h和12 h。数据为3个重复的平均值±标准偏差。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.05)。"

表4

方法A3B2C1D2提取4种激素稳定性及重复性结果"

激素
Hormone		 稳定性相对标准偏差
Stability relative standard deviation (RSD)		 重复性相对标准偏差
Repetitive relative standard deviation (RSD)
ABA 3.29 4.35
IAA 7.80 3.51
SA 4.36 4.32
JA 5.55 4.40

图4

优化方法与其他5种方法提取效果比较 缩写同图1。A~D依次是利用6种方法提取的E3叶片的IAA、ABA、SA、JA的含量。横坐标1~6依次对应表3中提取方法1-6, 6为优化方法。数据为3个重复的平均值±标准偏差。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.05)。"

图5

华1和中5植株中各部位激素含量 缩写同图1。A~D和E~H分别是华1和中5根、茎、叶、顶芽、匍匐茎中JA、IAA、SA、ABA含量, 数据为3个重复的平均值±标准偏差。不同字母表示由方差分析确定的各组间的统计学差异(Tukey多重比较, P < 0.01)。"

图6

华1块茎休眠过程中顶芽长度统计 A: 华1块茎在不同温度顶芽状态图, 比例尺长度为1.5 cm; B: 不同温度下华1块茎顶芽达到2 mm数目占总数的比例变化; C: 华1块茎顶芽达到5 mm数目占总数的比例变化。数据为3个重复的平均值±标准偏差。"

图7

中5块茎休眠过程中顶芽长度统计 A: 中5块茎在不同温度顶芽状态图, 比例尺长度为1.5 cm; B: 不同温度下中5块茎顶芽达到2 mm数目占总数的比例变化; C: 中5块茎顶芽达到5 mm数目占总数的比例变化。数据为3个重复的平均值±标准偏差。"

图8

华1和中5休眠期顶芽内源激素随时间变化检测结果 缩写同图1。A~C和D~F分别是华1和中5不同储存阶段的激素含量。数据为3个重复的平均值±标准偏差。"

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