毛果杨中与PtMKK4互作的PtMPKs的筛选及验证

doi:10.11707/j.1001-7488.20151008

Abstract

Abstract: [Objective] Mitogen-activated protein kinase (MAPK) cascades play central roles in the signal transduction networks from external stimuli to the intracellular responses. A typical MAPK cascade consists of three subsequently acting protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and finally, the MAP kinase (MAPK). Poplar is not only the model organism of forest research, but also the important economic tree species of China. Thus, it is of great significance to carry out research on the MAPK signal pathways of poplar under various stress conditions. To date, a complete MAPK signal pathway has not yet been established in poplar. PtMKK4 has been shown to be involved in mediating the drought signal in Populus trichocarpa. It will provide insights into the molecular mechanism of MAPK cascades regulating the plant stress responses to identify the downstream PtMPKs target of PtMKK4. [Method] In this study, yeast two-hybrid system and bimolecular fluorescence complementation(BiFC)were performed to screen and verify of the PtMPKs, which interacted with PtMKK4. Semi-quantitative RT-PCR was used to analyze the expression patterns of the identified PtMPKs under drought and high salinity conditions. [Result] pGBKT7-PtMKK4 bait plasmid and pGADT7-PtMPKs prey plasmids were constructed, respectively, and co-transformed into the yeast Y₂H competent cells. The results showed that only the Y₂H cells transformed with pGBKT7-PtMKK4 and pGADT7-PtMPK6-1 plasmids could grow in the nutrient deficient media SD/-Ade/-Trp/-Leu/-His and SD/-Ade/-Trp/-Leu/-His/X-α-gal, and furthermore the yeast colonies in the SD/-Ade/-Trp/-Leu/-His/X-α-gal medium were blue. The BiFC results also confirmed that PtMKK4 interacted with PtMPK6-1. Besides, the identified PtMPK6-1 could be induced by 20% PEG and 200 mmol·L^-1 NaCl, which was similar to PtMKK4. [Conclusion] These results above indicated that PtMPK6-1 may be the downstream target of PtMKK4 in P. trichocarpa.

Key words: Populus trichocarpa, MAPK cascades, yeast two-hybrid system, bimolecular fluorescence complementation

CLC Number:

S718.46

Wang Lei, Su Hongyan, Gu Liang, Chang Zhiyuan, Chen Na. Screening and Verification of PtMPKs Interacting with PtMKK4 of Populus trichocarpa[J]. Scientia Silvae Sinicae, 2015, 51(10): 60-66.

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Screening and Verification of PtMPKs Interacting with PtMKK4 of Populus trichocarpa

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