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作物学报 ›› 2023, Vol. 49 ›› Issue (9): 2594-2600.doi: 10.3724/SP.J.1006.2023.24225

• 研究简报 • 上一篇    

木薯蛋白激酶MeSnRK2.12与转录因子MebHLH1的互作鉴定及其表达分析

郁雪婷1(), 李可2, 李梦桃1, 鲍茹雪1, 陈新3,*(), 王文泉1   

  1. 1海南大学热带作物学院, 海南海口, 570228
    2海南大学生命科学学院, 海南海口 570228
    3中国热带农业科学院热带生物技术研究所 / 海南热带农业资源研究院海南省热带农业生物资源保护与利用重点实验室, 海南海口 571101
  • 收稿日期:2022-10-10 接受日期:2023-02-14 出版日期:2023-09-12 网络出版日期:2023-02-28
  • 通讯作者: *陈新, E-mail: chenxin@itbb.org.cn
  • 作者简介:郁雪婷, E-mail: yuxueting_2019@sina.com
  • 基金资助:
    国家重点研发计划项目(2018YFD1000500);国家自然科学基金项目(31861143005);海南省高校研究生创新科研课题(Hyb2019-10)

Interaction identification between protein kinase MeSnRK2.12 and transcription factor MebHLH1 and its relative expression level in cassava

YU Xue-Ting1(), LI Ke2, LI Meng-Tao1, BAO Ru-Xue1, CHEN Xin3,*(), WANG Wen-Quan1   

  1. 1College of Tropical Crops, Hainan University, Haikou 570228, Hainan, China
    2School of Life Sciences, Hainan University, Haikou 570228, Hainan, China
    3Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences / Hainan Key Laboratory of Conservation and Utilization of Tropical Agricultural Biological Resources, Hainan Institute of Tropical Agricultural Resources, Haikou 571101, Hainan, China
  • Received:2022-10-10 Accepted:2023-02-14 Published:2023-09-12 Published online:2023-02-28
  • Supported by:
    National Key Research and Development Program of China(2018YFD1000500);National Natural Science Foundation of China(31861143005);Innovative Research Projects for Graduate Students of Hainan Province(Hyb2019-10)

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摘要：

蛋白激酶SnRK2s (Sucrose Non-fermenting Related Protein Kinase 2)是植物抗逆境机制中的关键组分。木薯是全球重要的食品和工业作物, 具有高淀粉累积和耐逆境的特点。迄今对木薯MeSnRK2家族成员参与逆境下淀粉合成调控的内在机制尚不清楚。本文围绕SnRK2家族受ABA微弱诱导的成员MeSnRK2.12展开研究, 先对其进行生物信息学分析后发现其启动子区分布逆境响应元件: 干旱胁迫MBS和ABA应答ABRE等顺式作用元件, 且其氨基酸序列与AtSnRK2.8和OsSAPK1/2高度同源。ABA和PEG6000处理木薯SC8植株后发现, MeSnRK2.12可以在2 h内快速响应ABA和PEG6000处理, 其转录活性在根中被抑制; 在茎中被诱导上调, 最高值分别为对照的15.0倍和8.0倍; 在叶中也呈现上调趋势, 但程度低于茎中。亚细胞定位试验结果显示MeSnRK2.12分布于细胞质和细胞核, 利用酵母双杂交和双分子荧光互补(BiFC)试验均验证了MeSnRK2.12和转录因子MebHLH1间存在互作, 且前期研究发现MebHLH1可以上调木薯蔗糖合酶基因MeSus1的转录活性, 而蔗糖合酶的活性与植物库强直接相关。因此, 推测MeSnRK2.12不仅在木薯应对逆境胁迫中发挥具有作用, 还可能参与ABA信号介导的淀粉合成调控, 有助于木薯在逆境条件下获得相对较高的淀粉产量。

关键词: 木薯, SnRK2s, bHLH, ABA, PEG

Abstract:

Protein kinase SnRK2s (Sucrose Non-fermenting Related Protein Kinase 2) is a key component of plant stress resistance mechanism. Cassava is an important food and industrial crop in the world, with the characteristics of high starch accumulation and stresses resistance. So far, the mechanism of MeSnRK2 family member involved in the regulation of starch synthesis under stress is unclear. MeSnRK2.12 was chosen in this study, which is a member of the weakly ABA-induced SnRK2s. Firstly, some stress-response cis-elements distributed in its promoter region, such as drought stress cis-element MBS and ABA response element ABRE. Secondly, its amino acid sequence was highly homologous to AtSnRK2.8 and OsSAPK1/2. MeSnRK2.12 could respond quickly to ABA and PEG treatments within two hours, and its transcription activity was inhibited in roots, but was induced in stem, and the highest values were 15 times and 8 times of the control, respectively. There was also an significant up-regulation trend in leaves, but the degree was lower than that in stems. Subcellular localization showed that MeSnRK2.12 located in the cytoplasm and cell nucleus. The interaction between MeSnRK2.12 and MebHLH1 was verified by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments. Previous studies revealed that MebHLH1 could up-regulate the transcription activity of MeSus1, and the activity of sucrose synthase was directly related to plant sink strength. Therefore, it is speculated that MeSnRK2.12 not only plays an important role in response to stress, but also may be involved in the regulation of starch synthesis mediated by ABA signal, which helps cassava achieve relatively high starch yield under stress conditions.

Key words: cassava, SnRK2s, bHLH, ABA, PEG

表1

试验所用引物"

引物名称Primer name 引物序列Primer sequence (5°-3°) 用途Purpose
MeSnRK2.12 F CATATGGAGCGTTATGAGATTATCAAAG 全长扩增克隆Full length amplification
MeSnRK2.12 R ACTAGTCAAAGGGCATACGAAATC
Tubulin-F GTGGAGGAACTGGTTCTGGA 荧光定量PCR RT-qPCR
Tubulin-R TGCAACTCATCTGCATTTCTCC
qMeSnRK2.12 F GACGTTTGGTCTTGTGGAGT 荧光定量PCR RT-qPCR
qMeSnRK2.12 R GATAACAGGTGCCTGCATTCTA

图1

MeSnRK2.12、OsSAPK1、OsSAPK2和AtSnRK2.8氨基酸序列比对"

图2

MeSnRK2.12基因响应PEG胁迫的表达模式 R、S和L分别为PEG-6000处理下的根、茎和叶。柱上不同小写字母表示存在0.05概率水平差异显著。"

图3

MeSnRK2.12基因响应ABA处理的表达模式 R1、S1和L1分别为1.0 μmol L-1ABA处理下的根、茎和叶; R10、S10和L10分别为1.0 μmol L-1ABA处理下的根、茎和叶; R100、S100和L100分别为100.0 μmol L-1ABA处理下的根、茎和叶。柱上不同小写字母表示在0.05概率水平差异显著。"

图4

MeSnRK2.12亚细胞定位 标尺为20 μm。"

图5

酵母双杂交分析证明SnRK2.12与bHLH1互作"

图6

双分子荧光互补试验检测SnRK2.12与bHLH1互作 标尺为20 μm。"

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