Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (22): 4416-4430.doi: 10.3864/j.issn.0578-1752.2024.22.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Functional Verification of SiCIPK21 Gene in Foxtail Millet

DU YanWei1(), YAN XiaoGuang1(), ZHAO JinFeng1(), JIA SuQing2, WANG GaoHong1, YU AiLi1, ZHANG Peng1   

  1. 1 Millet Research Institute, Shanxi Agricultural University, Changzhi 046011, Shanxi
    2 Maize Research Institute, Shanxi Agricultural University, Xinzhou 034000, Shanxi
  • Received:2024-05-12 Accepted:2024-07-12 Online:2024-11-16 Published:2024-11-22
  • Contact: YAN XiaoGuang, ZHAO JinFeng

Abstract:

【Objective】The Ca2+-CBL-CIPK signaling pathway has important functions in plant response to abiotic stresses. By cloning the SiCIPK21 gene and studying its function under stress conditions, we provide a key candidate gene and theoretical basis for molecular breeding of foxtail millet with stress tolerance.【Method】Bioinformatics was used to analyze the cis-acting elements in the promoter region of this gene and predict the interactions between this protein and AtCBLs in Arabidopsis thaliana. SiCIPK21 was cloned by PCR, and a fusion expression vector was constructed for transient expression in tobacco to determine the subcellular localization. foxtail millet cv. Yugu 1 was used as material, and specifically amplified part of the SiCIPK21 gene fragment from Yugu 1 leaves, and recombinant vector VIGS-pTRV2-SiCIPK21 was constructed, using the phytoene desaturase gene (SiPDS) as the indicator gene, and seedlings of foxtail millet at the two-leaf stage were selected and infiltrated by cotyledon injection to investigate the role of SiCIPK21 under salt stress (250 mmol·L-1 NaCl) by using virus-induced gene silencing (VIGS) technology. T3 generation transgenic lines were obtained by overexpressing SiCIPK21 in Arabidopsis thaliana. Phenotypes at germination were analyzed under different concentrations of NaCl (150/175 mmol·L-1), mannitol (300/400 mmol·L-1) and ABA (0.25/0.5 μmol·L-1) treatments, and salt and drought tolerant phenotypes at seedling stage were also analyzed.【Result】Subcellular localization revealed that SiCIPK21 was located in the nucleus. The protein SiCIPK21 might interact with AtCBL2, AtCBL3, AtCBL4, AtCBL9, and AtCBL10 in Arabidopsis thaliana. The promoter region of SiCIPK21 contained adverse response elements, suggesting that SiCIPK21 may participate in the adverse responses. The VIGS gene silencing demonstrated that SiCIPK21-silenced foxtail millet plants had increased sensitivity to salt stress than the control plants. Three independent T3 generation Arabidopsis thaliana overexpression lines (2#, 3# and 6#) were obtained by genetic transformation. Overexpression lines showed significantly higher germination rate, germination speed, green cotyledon unfolding rate, root length and fresh weight than the wild-type plants (WT) at different concentrations of NaCl (150/175 mmol·L-1), mannitol (300/400 mmol·L-1) and ABA (0.25/0.5 μmol·L-1). Moreover, phenotypic analysis of salt and drought tolerance in Arabidopsis seedlings showed that overexpression lines had significantly higher survival rates and chlorophyll contents than WT.【Conclusion】SiCIPK21 is a positive regulator of plant response to salt and drought stresses, which makes it a candidate gene for improving stress tolerance by molecular breeding in foxtail millet.

Key words: foxtail millet, SiCIPK21, abiotic stress, phenotype analysis, gene silencing

Table 1

Primer sequences used in this study"

引物名称 Primer name 引物序列 Primer sequence (5′-3′)
SiCIPK21-GFP-F ACTAGGGTCTCGCACCATGGCCATGGAGAAGAACCAAGACA
SiCIPK21-GFP-R ACTAGGGTCTCTCGCCCGTGGTGGTCCGCCTCG
VIGS-pTRV2-SiCIPK21-F AAGGTTACCGAATTCTCTAGGTTAGTGAAAAGGGGATCCTCATTTCCTC
VIGS-pTRV2-SiCIPK21-R GAGACGCGTGAGCTCGGTACGAAATTCTCCAGGTGATCATGGGTGTG
pCMBIA3301-SiCIPK21-F ACTAGGGTCTCGCACCATGGCCATGGAGAAGAACCAAGACA
pCMBIA3301-SiCIPK21-R ACTAGGGTCTCTACCGCTACGTGGTGGTCCGCCTC
RT-qPCR-SiCIPK21-F TAATCGCCAAGCACCTCCTA
RT-qPCR-SiCIPK21-R CTTGGTTCTTCTCCATGGCC
PCR-SiCIPK21-F ATGGCCATGGAGAAGAACCAAGACA
PCR-SiCIPK21-R CCTGTAGTTGCTCTGCGTGA
β-Actin-F CAGTGGACGCACAACAGGTAT
β-Actin-R AGCAAGGTCAAGACGGAGAAT

Fig. 1

Subcellular localization of SiCIPK21 in tobacco epidermis"

Table 2

Prediction of cis-elements in the promoter region of SiCIPK21 gene"

顺式元件Cis-element 典型序列Typical sequence 特性Characteristic
TGA-element AACGAC 生长素响应Auxin-responsive element
P-box CCTTTTG 赤霉素响应Gibberellin-responsive element
CGTCA-motif CGTCA 茉莉酸甲酯响应Cis-acting regulatory element involved in the MeJA-responsiveness
TGACG-motif TGACG
LTR CCGAAA 低温响应Cis-acting element involved in low-temperature responsiveness
MBS CAACTG 干旱响应MYB binding site involved in drought-inducibility
ARE AAACCA 厌氧响应Cis-acting regulatory element essential for the anaerobic induction
ATCT-motif AATCTAATCC 光响应Part of a conserved DNA module involved in light responsiveness
Box 4 ATTAAT
GATA-motif GATAGGA 光响应Part of a light responsive element
TCT-motif TCTTAC
chs-CMA2a TCACTTGA
AE-box AGAAACAA
MSA-like TCAAACGGT 细胞周期调控Cis-acting element involved in cell cycle regulation
TC-rich repeats ATTCTCTAAC 防御和应激反应响应Cis-acting element involved in defense and stress responsiveness

Fig. 2

Construction of VIGS vector of SiCIPK21 and characterization the salt tolerance of silencing SiCIPK21 in foxtail millet A: Construction of VIGS vector of SiCIPK21. a: PCR product amplification of SiCIPK21 fragment; b: Restriction digestion verification of pTRV2-SiCIPK21 vector. M: DNA Marker. B: Phenotypes of silenced SiPDS and SiCIPK21 in foxtail millet plants. a: Phenotype of control (pTRV2-00) and SiPDS-silenced plants (pTRV2-SiPDS) (The left is the control and the right is the SiPDS silent plant); b. Phenotypes of control (pTRV2-00) and SiCIPK21-silenced (pTRV2-SiCIPK21) plants under salt stress (250 mmol·L-1 NaCl) (The left is the control and the right is the SiCIPK21 silent plant); c: Phenotype of SiPDS-silenced plants. C: SiCIPK21 gene silencing efficiency detection. D: Chlorophyll content in leaves of control and SiCIPK21 silent plants under salt stress. E: MDA content in leaves of control and SiCIPK21 silent plants under salt stress. * and ** indicate significant difference at the 0.05 and 0.01 probability levels, respectively. The same as below"

Fig. 3

Identification of SiCIPK21 overexpression in Arabidopsis A: SiCIPK21 was detected by agarose gel electrophoresis; M: DNA marker DL2000. B: PCR detection of SiCIPK21 overexpression in Arabidopsis plants; 1, 2, 3, 6, 8, 10, 11, 12: Plants of SiCIPK21 overexpression, -: Negative control, +: Positive control. C: Relative expression levels of SiCIPK21 in Arabidopsis plants. *** indicates that the difference is extremely significant at the 0.001 probability levels"

Fig. 4

Phenotypes of Arabidopsis overexpressing SiCIPK21 under salt stress at germination stage A, B: Phenotype and opening cotyledon rate of overexpression Arabidopsis lines (2#, 3# and 6#) after treatment with 0, 150 and 175 mmol·L-1 NaCl for 12 d; C: Germination rate of overexpression Arabidopsis lines (2#, 3# and 6#) after treatment with 150 mmol·L-1 NaCl for 7 d; D-F: Phenotype, fresh weight and root length of overexpression Arabidopsis lines (2#, 3# and 6#) measured at 12 d after treatment with 0, 150 and 175 mmol·L-1 NaCl"

Fig. 5

Effect of seedling salt stress on the growth of SiCIPK21 overexpressing Arabidopsis A: Photographs of overexpression Arabidopsis lines (2#, 3# and 6#) before and after salt stress (200 mmol·L-1 NaCl); B: Statistics of survival rates; C: Chlorophyll content of overexpression Arabidopsis lines (2#, 3# and 6#) treated with NaCl"

Fig. 6

Osmotic tolerance of SiCIPK21 overexpressing Arabidopsis at germination stage A, B: Phenotype and opening cotyledon rate of overexpression Arabidopsis lines (2#, 3# and 6#) after treatment with 0, 300 and 400 mmol·L-1 mannitol for 12 d; C: Germination rate of overexpression Arabidopsis lines (2#, 3# and 6#) after treatment with 300 mmol·L-1 mannitol for 7 d; D-F: Phenotype, fresh weight and root length of overexpression Arabidopsis lines (2#, 3# and 6#) measured at 12 d after the treatment with 0, 300 and 400 mmol·L-1 mannitol"

Fig. 7

Effect of drought stress on the growth of seedlings overexpressing SiCIPK21 Arabidopsis A: Photographs of overexpression Arabidopsis lines (2#, 3# and 6#) before and after drought treatment; B: Statistics of survival rates; C: Chlorophyll content of overexpression Arabidopsis lines (2#, 3# and 6#) treated with drought"

Fig. 8

ABA tolerance in SiCIPK21 overexpressing Arabidopsis plants at germination stage A, B: Phenotype and opening cotyledon rate of overexpression Arabidopsis lines (2#, 3# and 6#) after treatment with ABA 0, 0.25 and 0.5 μmol·L-1 ABA for 12 d; C: Germination rate of overexpression Arabidopsis lines (2#, 3# and 6#) treatment with 0.25 μmol·L-1 ABA for 7 d; D-F: Phenotype, fresh weight and root length of overexpression Arabidopsis lines (2#, 3# and 6#) measured at 12 d after the treatment with 0, 0.25 and 0.5 μmol·L-1 ABA"

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