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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (5): 1272-1281.doi: 10.3724/SP.J.1006.2023.24070

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

GmCIPK10, a CBL-interacting protein kinase promotes salt tolerance in soybean

LI Hui1,2(), LU Yi-Ping1, WANG Xiao-Kai1, WANG Lu-Yao1, QIU Ting-Ting1, ZHANG Xue-Ting1, HUANG Hai-Yan1, CUI Xiao-Yu1,*()   

  1. 1College of Agriculture and Forestry Sciences, Linyi University, Linyi 276000, Shandong, China
    2Center for International Education, Philippine Christian University, 1004, the Philippines
  • Received:2022-03-29 Accepted:2022-07-21 Online:2023-05-12 Published:2022-08-18
  • Contact: *E-mail: cuixiaoyu@lyu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32001459);Natural Science Foundation of Shandong Province(ZR2020QC123)

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Abstract:

Salt stress seriously restricts the yield and quality of soybean. Calcineurin B subunit-interacting protein kinases (CIPKs) play a vital role in response to environmental stresses in plant. However, few study is known about the biological function of soybean CIPKs. In this study, GmCIPK10 was cloned from soybean genome. Bioinformatics analysis exhibited that GmCIPK10 encoded an intron-poor type CIPKs with a serine (Ser)/threonine (Thr) protein kinase domain and a NAF/FISL motif. The relative expression pattern levels showed that the transcript level of GmCIPK10 was increased under NaCl, MV, and H2O2 treatments. Overexpression of GmCIPK10 in Arabidopsis and soybean hairy roots improved salt tolerance of transgenic plants. Further physiological indicator assays demonstrated that GmCIPK10 overexpression could decrease the accumulation of MDA and H2O2, enhance the activity of antioxidant enzymes, and reduce sodium (Na+)/potassium (K+) in transgenic plants under salt stress. In addition, qRT-PCR illustrated that GmCIPK10 promoted the relative expression level of antioxidant- and salt tolerance-related gene in salt stress. The yeast two-hybrid, pull-down, and bimolecular fluorescence complementation experiments confirmed that GmCIPK10 interacts with the Ca2+ sensor GmCBL4. These results provide a reference for investigating the role of the CBL-CIPK signaling pathway in response to salt stress in soybean.

Key words: GmCIPK10, salt tolerance, ROS scavenging, Na+/K+ homeostasis, soybean

Table 1

Primers used in this study"

引物
Primer ID		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
AtActin GAAATCACAGCACTTGCACC TGGAATGTGCTGAGGGAAGC
GmTubulin GAGGCAAGATGAGCACCAAG ACGGAACATTTCCTGAATGGAG
GmCIPK10 CAAGCCTGTTTTTGCATCTGAAT GATAGTGTGTTTGGATCCCAGC
GmCAT1 AGCTAGCGCAAAGGGTTTCT AAGGTTTCAGGGCTACCACG
GmPOD21 CCGTTTCGTGGGTCAGAAATCT- CCGACGCCTGCTCCGACACTA
GmZAT10 AAGCCTTCTCCTCTTACCAAGCA TCGACGCCGA ACTCGTTGT
GmMYB118 ATCATACTGTTCGGAGTCAC CAGACACTGTAGAGACCTTGTT
GmLEA5 CCGATGTATCGGTAAGAGT AGGCTTTTGA ACCATCTC
GmNHX1 GTCGGGGCACACTTCACTAA GGATGCTGCTTGGACGATGA
GmSALT3 AAGCAGGTGCTTAACGACGA CAAATCTGTTAGCCGCGACG
GmSOS1 ATCGGCTGGGAAAGATTGGG CACCAGGGCCAGCTAGTAAG

Fig. 1

Protein structure and evolutionary relationship of GmCIPK10 protein"

Fig. 2

Relative expression profiles of GmCIPK10 in salt and oxidative stresses GmTubulin is used as an internal control; each group contains three seedlings for three biological replicates. MV: methyl viologen. *: P < 0.05."

Fig. 3

Overexpression of GmCIPK10 enhances salt tolerance ability in transgenic Arabidopsis A, B: the phenotypes and primary root length of GmCIPK10-OE and WT Arabidopsis plants with salt treatment (75 mmol L-1 NaCl) for 10 days; C: the expression level of GmCIPK10 genes; D: MDA content; E: H2O2 content; Each group contained four seedlings for three biological replicates; WT: wild type; OE: overexpression. *: P < 0.05."

Fig. 4

Overexpression of GmCIPK10 enhances salt tolerance ability in transgenic soybean hairy roots A, B: the phenotype and survival rate of GmCIPK10-OE and VC soybean plants with salt treatment (200 mmol L-1 NaCl) for 10 days; C: the expression level of GmCIPK10 genes; D: MDA content; E: H2O2 content; F: CAT activity; G: POD activity; H: K+ content; I: Na+ content; J: Na+/K+ ratio of GmCIPK10-OE and VC soybean plants with salt treatment for 7 days; VC: vector control; OE: overexpression; FW: fresh weight; DW: drought weight. *: P < 0.05."

Fig. 5

Relative expression level of GmCIPK10 genes in salt and oxidative stresses Each group contained four seedlings for three biological replicates. VC: vector control; OE: overexpression. *: P < 0.05."

Fig. 6

GmCBL4 interacts with GmCIPK10 A: yeast two-hybrid analysis of GmCBL4 interaction with GmCIPK10; B: pull-down assays of the interaction of GmCBL4 with GmCIPK10; C: BiFC assays of the interaction of GmCBL4 with GmCIPK10. BiFC: bimolecular fluorescence complementation; YFP: yellow fluorescent protein. Bar: 12 μm."

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