Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (19): 3697-3709.doi: 10.3864/j.issn.0578-1752.2022.19.002

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

Cloning and Functional Analyses of MsCIPK2 in Medicago sativa

SU Qian1,2(),DU WenXuan1,MA Lin1,XIA YaYing1,LI Xue1,QI Zhi2(),PANG YongZhen1()   

  1. 1Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193
    2School of Life Sciences, Inner Mongolia University, Hohhot 010021
  • Received:2022-04-21 Accepted:2022-06-02 Online:2022-10-01 Published:2022-10-10
  • Contact: Zhi QI,YongZhen PANG E-mail:2646335315@qq.com;qizhi@imu.edu.cn;pangyongzhen@caas.cn

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Abstract:

【Objective】 CIPKs are a group of important protein kinase involved in signaling pathway of plant in response to stress. They can form CBL-CIPK complex with CBL, to activate the expression of related responsive genes to cope with various abiotic stresses in cells. Exploration and study on the molecular mechanism of MsCIPK genes in alfalfa in response to abiotic stress will help to reveal the biological basis of stress resistance in alfalfa, and to provide new gene resources for alfalfa breeding with enhanced stress resistance. 【Method】 The MsCIPK2 gene was cloned by using PCR, the sequence was analyzed by bioinformatics tools, and the expression level of MsCIPK2, MsCBL2, MsCBL6, MsCBL7 and MsCBL10 genes in various tissues were analyzed by using qRT-PCR. The pCAMBIA1302-GFP-MsCIPK2 vector was transiently expressed in tobacco leaf epidermal cells, and the subcellular localization was observed under laser confocal microscope. Yeast two-hybrid assay was used to analyze interaction between MsCIPK2 and four MsCBLs proteins. Agrobacterium rhizogenes was used to generate alfalfa hairy roots over-expressing MsCIPK2. qRT-PCRs were used to analyze the expression levels of related genes in transgenic hairy root lines. 【Result】 The coding sequence of MsCIPK2 gene was obtained by using PCR, and it is 1 230 bp in length, encoding 409 amino acids. The deduced MsCIPK2 protein contained typical ATP binding site, activation loop, NAF motif and PPI motif as for the CIPK family genes. The expression level of MsCIPK2 gene was the highest in roots, and the lowest in the flowers of alfalfa. Subcellular localization results showed that MsCIPK2 protein was localized in the endoplasmic reticulum. Yeast two-hybrid assays showed that MsCIPK2 protein interacted with MsCBL2, MsCBL6, MsCBL7 and MsCBL10 proteins, showing stronger interaction with MsCBL10 than with other MsCBLs. The expression levels of MsCBL2, MsCBL6, and MsCBL10 were the highest in roots of alfalfa, and the expression level of MsCBL7 was the highest in pods. qRT-PCR results showed that the expression levels of abiotic stress-associated genes ATPase, P5CS, CYP705A5, COR47, HAK5 and RD2 were significantly up-regulated in hairy roots over-expressing MsCIPK2. Under the treatment of 200 mmol·L-1 NaCl and 20% PEG, when compared with the control hairy root line, hairy roots over-expressing MsCIPK2 had lower MDA content, and higher POD activity, proline content and soluble sugar content. 【Conclusion】 MsCIPK2 can interact with CBL protein, and responded to salt and drought stress in roots of alfalfa. Over-expression of MsCIPK2 can improve salt and drought stress resistance in alfalfa, and MsCIPK2 can be used as candidate gene for alfalfa breeding with improved abiotic stress resistance.

Key words: Alfalfa, CIPK, CBL, abiotic stress

Table 1

Primer sequence used in this study"

引物名称 Primer name 序列 Sequences (5′-3′)
MsCIPK2-F ATGGGAAGAGTACTAGGTAAAG
MsCIPK2-R TCACTCCCCTTGCCATGACC
MsCIPK2-RT-PCR-F TGCCGTAGATTATTGCCACAGTAG
MsCIPK2-RT-PCR-R ACACCACAAGACCAAGTATCAGC
MsCBL2-RT-PCR-F CGTTGTGGTGATGTCCTTGAG
MsCBL2-RT-PCR-R CGGAGTCTGGTATGAATCTATCG
MsCBL6-RT-PCR-F GTTGCTACGCTTGCTGAATCTG
MsCBL6-RT-PCR-R ATGTTCTTGAGTAATGATGGATGGC
MsCBL7-RT-PCR-F TTTAGAAACAGAAACAAACGCAACC
MsCBL7-RT-PCR-R ATCATCAACACCATATCCTTCAACTC
MsCBL10-RT-PCR-F TCAAGTATCCCATTGTGCTTTGTATC
MsCBL10-RT-PCR-R AGAGAGACCGTGTTTAGTGTAAGTG
MsCIPK2-GFP-F AGAACACGGGGGACTCTTGACCATGGGAAGAGTACTAGGTAAAG
MsCIPK2-GFP-R GTGAAAAGTTCTTCTCCTTTACTAGTCTCCCCTTGCCATGACC
MsCBL2-yBD-F CTGATCTCAGAGGAGGACCTGCATATGATGGAATACAAAGCTTGGG
MsCBL2-yBD-R GCAGGTCGACGGATCCCCGGGAATTCTTAGAGGGATTCAAGTCTTTACTC
MsCBL6-yBD-F CTGATCTCAGAGGAGGACCTGCATATGATGTTGCAGTGCATAGAGGG
MsCBL6-yBD-R GCAGGTCGACGGATCCCCGGGAATTCTCAAGTATCGTCTACTTGTG
MsCBL7-yBD-F CTGATCTCAGAGGAGGACCTGCATATGATGGGTTGCAGTAGCTCAAAAG
MsCBL7-yBD-R GCAGGTCGACGGATCCCCGGGAATTCCTACGATTGTGATTCTTCGGTTTC
MsCBL10-yBD-F CTGATCTCAGAGGAGGACCTGCATATGATGCTTGTTATCGTATTCGCG
MsCBL10-yBD-R GCAGGTCGACGGATCCCCGGGAATTCTCAGGCTGACGCGACATCGAC
MsCIPK2-yAD-F CGACGTACCAGATTACGCTCATATGATGGGAAGAGTACTAGGTAAAG
MsCIPK2-yAD-R CGTATCGATGCCCACCCGGGTGGAATTCTCACTCCCCTTGCCATGACC

Fig. 1

Analysis of MsCIPK2 protein sequence A: Prediction of the hydrophobicity; B: Prediction of the secondary structure; C: Protein sequences alignment of MsCIPK2 with CIPKs of different plant species. TcCIPK25: Theobroma cacao, EOY29347.1; DzCIPK25: Durio zibethinus, XP_022730333.1; HuCIPK25: Herrania umbratica, XP_021276011.1; ApCIPK5: Abrus precatorius, XP_027354272.1; MtCIPK25: Medicago truncatula, XP_003588823.1; TpCIPK25: Trifolium pratense, XP_045823300.1; CaCIPK25: Cicer arietinum, XP_004498818.1"

Fig. 2

Tissue-specific expression of MsCIPK2 gene * indicates significant difference with P<0.05, ** indicates highly significant difference with P<0.01. The same as below"

Fig. 3

Subcellular localization of MsCIPK2 protein"

Fig. 4

Yeast two-hybrid assays for MsCIPK2 and four MsCBLs proteins"

Fig. 5

Tissue-specific expression of four MsCBLs"

Fig. 6

Identification of alfalfa hairy roots over-expressing MsCIPK2 A, B: Transformation of hairy roots over-expressing MsCIPK2; C: Identification of hairy roots over-expressing MsCIPK2 by using PCR; D: Detection of gene expression level in MsCIPK2 over-expression hairy root"

Fig. 7

Expression levels of stress-related genes in hairy roots over-expressing MsCIPK2"

Fig. 8

Effects of physiological indicators in transgenic and the control hairy roots under stress treatments A: MDA content under 200 mmol·L-1 NaCl treatment; B: SOD activity under 200 mmol·L-1 NaCl treatment; C: Proline content under 200 mmol·L-1 NaCl treatment; D: Soluble sugar content under 200 mmol·L-1 NaCl treatment; E: MDA content under 20% PEG treatment; F: SOD activity under 20% PEG treatment; G: Proline content under 20% PEG treatment; H: Soluble sugar content under 20% PEG treatment"

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