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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (01): 20-30.doi: 10.3724/SP.J.1006.2020.92007


Overexpression of OsMPK17 protein enhances drought tolerance of rice

MA Jin-Jiao1,LAN Jin-Ping1,2,ZHANG Tong1,CHEN Yue1,GUO Ya-Lu1,3,LIU Yu-Qing1,YAN Gao-Wei1,WEI Jian1,DOU Shi-Juan1,YANG Ming1,LI Li-Yun1,LIU Guo-Zhen1,*()   

  1. 1 College of Life Sciences, Hebei Agricultural University, Baoding 071001, Hebei, China
    2 Research Center for Life Sciences, Hebei North University, Zhangjiakou 075000, Hebei, China
    3 Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518116, Guangdong, China
  • Received:2019-02-26 Accepted:2019-08-09 Online:2020-01-12 Published:2019-09-04
  • Contact: Guo-Zhen LIU E-mail:gzhliu@hebau.edu.cn
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31171528)


Mitogen-activated protein kinase (MAPK) highly conserved in eukaryotes plays important roles in stress responses in plant. In this study, full-length OsMPK17 gene was cloned and fusion protein was expressed. The purified protein was used as immunogen to generate monoclonal antibody. Western blot (WB) analyses were carried out for protein samples isolated from tissues under different abiotic stresses. The expression of OsMPK17 was induced by drought stress, suggesting that the OsMPK17 protein may play a role in drought stress response. Proteins isolated from leaves treated with abscisic acid (ABA) or methylene jasminate acid (MeJA) demonstrated a decrease of OsMPK17 protein abundance, suggesting that hormones may be involved in the function of the protein. The overexpression vector of OsMPK17 protein was established and transformed into TP309 via Agrobacteria-mediated protocol. Homozygous transgenic lines for overexpression of OsMPK17 protein were obtained. In the field planting experiment, the plant height and the spike length of transgenic lines shortened and the seed setting rate decreased. At seed germination stage, under the condition of PEG-6000 treatment, the seeds of overexpressed OsMPK17 protein lines grew better and the length of root and shoot was significantly longer than those of the wild type. At seedling stage, transgenic plants showed lower water loss rate when exposed in the air. The transgenic rice with overexpressed OsMPK17 protein grew better than the wild type in the experiment with soil drought stress and re-watering then. In conclusion, the overexpressed OsMPK17 protein enhances drought tolerance of rice. This study enhances the understanding for the function of OsMPK17 protein.

Key words: rice, MAPK protein, Western blot, stress, overexpression, antibody-based proteomics

Fig. 1

Cloning of rice OsMPK17 gene and fusion protein expression A: PCR amplification of rice OsMPK17 gene. A plasmid containing full-length OsMPK17 gene was used as template for PCR amplification of OsMPK17 gene using primers 5′-GCGGTACCATGGG CGGCCGCGCCCGCTC-3′ and 5′-GCGAGCTCGGTTTTCAGTT GAGCAAC-3′. B: Verification of recombinant pET30a-MPK17 plasmid by double digestion using Kpn I and Sac I. The PCR products and pET30a plasmid DNA were digested by Kpn I and Sac I, the ligation product was used to transform DH5α. Recombinant plasmid was verified by double digestion. C: Induction of fusion protein OsMPK17 and Coomassie blue staining. Correct pET30a- MPK17 plasmid verified by double digestion was double checked by sequencing. Sequencing verified plasmid was transformed to Codon plus bacterial strain to express fusion protein. The bacteria was cultured in LB medium containing 50 μg mL-1 kanamycin and IPTG which was added when the OD600 reached 0.6-0.8. The bacteria was collected after over night culture at 25°C and disrupted by sonication. The supernatant (S) and pellet (P) were obtained after centrifugation and total protein was separated by 10% SDS-PAGE and stained with Coomassie blue. 0: Total protein isolated at 0 time point. M: Molecular weight marker; PCR: Amplification products; K+S: Double digestion product using Kpn I and Sac I."

Fig. 2

Expression profiling of OsMPK17 protein in rice by western blot analysis A: Drought stress treatment: rice seedlings grown for 5 days were treated by 20% PEG-6000. Leaf samples were collected at 0, 1 h, 2 h, 4 h, 8 h, 12 h, 1 d, 2 d, and 3 d respectively; WB analysis were carried out for isolated total proteins. HSP: Loading control for WB analysis using HSP82 antibody. B: Treatment with hormones: leaves of rice were cultured in petri dish, 100 μmol L-1 ABA or 100 μmol L-1 MeJA was supplemented as hormone treatments. Samples were collected at 0, 6 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, and 6 d time points, respectively. Total proteins were isolated and analyzed by WB. HSP: Loading control for WB analysis using HSP82 antibody."

Fig. 3

Construction and identification of rice OsMPK17 overexpression vector A: PCR amplification of rice OsMPK17 gene; B: Hind III+ Xba I restriction enzyme digestion of recombinant pEASY-MPK17-3HA plasmid; C: Kpn I+ Spel I restriction enzyme digestion of recombinant pUBI-C4300-MPK17 plasmid. PCR amplification of OsMPK17 gene using plasmid containing full-length OsMPK17 cDNA as template, the primers used were 5′-GCGGTACCATGGGC GGCCGCGCCCGCTC-3′ (Kpn I restriction site was underlined) and 5′-GCGAGCTCGGTTTTCAGTTGAGCAAC-3′ (Sac I restriction site was underlined). The amplified fragment was inserted into pEASY-3HA vector and verified by double digestion. Sequence verified pEASY-MPK17-3HA was digested by Kpn I+ Spe I, the fragment was inserted into pUBI-C4300 and verified by double digestion. M: Molecular weight marker; PCR: PCR amplification product; H+X: Hind III+ Xba I restriction enzyme digestion; K+S: Kpn I+ Spel I restriction enzyme digestion."

Fig. 4

Identification of transgenic rice plants with overexpression OsMPK17 protein Upper panel: PCR product; Middle panel: WB detection of OsMPK17 protein in transgenic rice plants; Lower panel: HSP signal was used as loading control; WT: wildtype rice plants; A202, A204, A210, and A212 are independent transgenic lines; 1, 2, 3, 4, 5, 6, 7, 8, and 9 are independent plants among the same transgenic lines; PCR: PCR products; MPK17-OX: Over expressed OsMPK17 protein; MPK17-Native: the original form of OsMPK17 protein in rice."

Fig. 5

Effects of over-expressed OsMPK17 protein on the phenotype and agronomic traits of rice Photographs on the upper panel: rice whole plants and ears at mature stage of four transgenic lines and control. Bar graphs on the lower panel: plant height, spike length, seed setting rate, and tillers number of the four transgenic lines and control."

Fig. 6

Characterization of the germination trait of rice seeds with over-expressed OsMPK17 protein under drought stress A: Photographs for seeds at germination. Upper panel: control (H2O); Lower panel: drought stress (20% PEG-6000) treatment. B: Bar graphs of root and shoot lengths for seeds at germination. WT: wild type; A202, A204, A210, and A212 are transgenic lines. Experiments were carried out with three replicates; average and standard derivations were calculated. * Significant at P < 0.05. ** Significant at P < 0.01."

Fig. 7

Characterization of water loss rate of OsMPK17 protein overexpressed transgenic lines WT: wild type; A202, A204, A210, and A212 are transgenic lines. At four leaves stage, leaf blades were cut into pieces at about 3 cm, which were weighed every 30 min at room temperature (30°C). The experiment were repeated three times; the average and standard derivation were calculated."

Fig. 8

Verification of drought tolerance at seedling stage of transgenic plants over-expressed OsMPK17 protein WT: wild type; A202 and A212 were transgenic lines overexpressed OsMPK17 protein. The drought and restore experiments were carried out at seedling stage; the photographs were taken at 0 time point, eight days after drought treatment, and re-watering for 3 days."

Supplementary table 1

Transcriptional abundance comparison of OsMPK17 gene among different rice tissues"

组织 Libraries FPKM
四叶期幼苗 Seedling four-leaf stage 0.640
幼苗地上部 Shoots 0.904
20 d的叶片 20-day leaves 0.637
抽穗前花序 Pre-emergence inflorescence 3.036
抽穗后花序 Post-emergence inflorescence 6.281
花药 Anther 169.643
雌蕊 Pistil 2.371
开花后5 d种子 5 DAP seed 5.971
开花后10 d种子 10 DAP seed 10.372
开花后25 d幼胚 25 DAP embryo 7.719
开花后25 d胚乳 25 DAP endosperm 7.230
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