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| Expression Analyses of Phosphoribulokinase Gene Induced by Stresses and Its Interacting Protein Screening in Rice (Oryza sativa) |
| HE Wei1,2,*, QU Meng-Yu1,2,*, WEI Yi-Dong1,2, LIAN Ling1,2, CAI Qiu-Hua1,2, ZHENG Yan-Mei1,2, WANG Ying-Heng1,2, ZHU Yong-Sheng1,2, XIE Hua-An1,2, ZHANG Jian-Fu1,2,** |
1 Rice Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350019, China;
2 State Key Laboratory for Ecological Control of Crop Pests between Fujian and Taiwan/National Engineering Laboratory of Rice/South China Research Base of State Key Laboratory of Hybrid Rice/Incubating base of State Key Laboratory of Crop Germplasm Innovation and Molecular Breeding between Fujian Province and Ministry of Science and Technology/Fuzhou Branch of National Rice Improvement Center/South China Key Laboratory of Hybrid Rice Germplasm innovation and Molecular Breeding of Ministry of Agriculture and Rural Areas/Fujian Engineering Laboratory of Crop Molecular Breeding/Fujian Key Laboratory of Rice Molecular Breeding, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China |
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Abstract Phosphoribulokinase (PRK) is a key enzyme in Calvin-Benson cycle which influences on plant growth and development. Nuclear-encoded plastid-localized PRK, coupling with other regulatory enzyme,involved in carbon assimilation in functional leaves. However, the research on RPK functions in stress response was still limited, especially in rice (Oryza sativa). In this study, target protein subcellular localization, expression characteristics analyses of rice phosphoribulokinase (OsPRK, GenBank No. LOC4330413) under abiotic stresses (drought, high salt and high temperature) and exogenous hormone treatments (abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA)), as well as MBP pull-down protein interaction screening were conducted to investigate the function of OsPRK involving in stress responses. By detecting the GFP report using laser confocal microscopy, the transient expression analysis demonstrated that OsPRK was strictly localized in chloroplast stroma of transformed rice protoplasts after 16 h protoplast culture, contrasting to control native GFP only found around the cytoplasm of protoplast. To characterize the change of the transcript abundance of OsPRK, quantitative realtime PCR (qPCR) was performed and found that the transcriptional expression of OsPRK was significantly inhibited at 0.5 and 3 h drought treatments, 1 and 6 h high salt or high temperature treatments, which implicated that OsPRK inclined to reduce transcriptional expression in drought, high salt and high temperature conditions. Beyond that, qPCR results also showed that the OsPRK transcription levels of seedlings under ABA and JA treatments were not significantly changed within 1 h, while continuously decreased when treated more than 4 h. Unlike ABA and JA treatment, the transcription level of OsPRK of SA treatment had a dramatical reduction from 0 to 8 h, and then gradually declined until 24 h. To investigate the potential OsPRK interacting proteins, OsPRK was expressed with MBP-Tag using recombinant plasmid pMAL-c5x and purified from BL21(DE3)plys prokaryotic expression system for pull-down protein interaction screening. The protein interaction experiment totally obtained 82 candidate proteins that might interact with OsPRK using MBP pull-down method. Of these, the functions of 67 candidate proteins were unknown while 15 candidate proteins were with known functions, including GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and WRKY transcription factor. This study found that OsPRK should be involved in abiotic stress and hormone response of rice seedlings. In addition, the study identified numbers of potential OsPRK interacting proteins, which can be used for further biological functions of OsPRK in stress avoidance and tolerance. All these findings accelerated the understanding of OsPRK roles in response to developmental requirements and environmental constraints.
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Received: 30 December 2021
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Corresponding Authors:
**jianfzhang@163.com
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| About author:: * These authors contributed equally to this work |
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