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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (9): 2594-2600.doi: 10.3724/SP.J.1006.2023.24225

• RESEARCH NOTES • Previous Articles    

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 Online:2023-09-12 Published: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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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

Table 1

Primers used in the study"

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

Fig. 1

Multiple sequence alignment for amino acid sequence of MeSnRK2.12, OsSAPK1, OsSAPK2, and AtSnRK2.8"

Fig. 2

Relative expression profile of MeSnRK2.12 genes under the infection of PEG stress R, S, and L represent roots, stems, and leaves were treated with PEG6000, respectively. Different lowercase letters on the column mean significant difference at the 0.05 probability level."

Fig. 3

Relative expression pattern of MeSnRK2.12 genes under ABA treatment R1/S1/L1: roots, stems, and leaves were treated with 1.0 μmol L-1 ABA, respectively; R10/S10/L10: roots, stems, and leaves were treated with 10.0 μmol L-1 ABA, respectively; R100/S100/L100: roots, stems, and leaves were treated with 100.0 μmol L-1 ABA, respectively. Different lowercase letters on the column mean significant difference at the 0.05 probability level."

Fig. 4

Subcellular localization of MeSnRK2.12 Bar: 20 μm."

Fig. 5

Interaction validation between SnRK2.12 and bHLH1 by Y2H assay in yeast cells"

Fig. 6

Interaction validation between SnRK2.12 and bHLH1 by bimolecular fluorescence complementation in onion epidemal cells Bar: 20 μm."

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