|
|
|
| Interaction Analysis of TaNRX1 with TaPDI, TaTRX-h and TaPP2Ac and Drought Resistance Study |
| TIAN Shu-Yuan, CHENG Jie, YANG Jie, LI Rui-Bo, ZHANG Yun-Rui, HAO Xi-Ying, GUO Yang-Yang, ZHANG Xiao-Ke* |
| College of Agronomy, Northwest A&F University, Yangling 712100, China |
|
|
|
|
Abstract Thioredoxin (TRX) is a kind of functionally diverse proteins with disulfide active sites, and plays an important role in many aspects including plant stress response. Nucleotoreoxin (NRX), a novel member of the TRX superfamily, was found to be associated with drought resistance in wheat (Triticum aestivum), but the mechanism of drought resistance is still unclear. Exploring the interaction protein of TaNRX1 will be helpful to further reveal the drought-resistant mechanism of TaNRX1. Based on previous studies of other researchers, the protein phosphatase 2A catalytic subunit (PP2Ac) was selected as candidate interaction protein. And according to bioinformatics analysis in STRING website (https://string-db.org/), other 2 proteins, protein disulfide isomerase (PDI) and TRX-h were also selected as TaNRX1-D candidate interaction proteins in this study. Then, the ORFs of TaPDI-A, TaPDI-B, TaPDI-D, TaTRX-h-A, TaPP2Ac-B, and TaPP2Ac-D were acquired by homologous cloning from drought-resistant cultivar 'Jinmai 47'. Furthermore, the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) were used to verify the interaction proteins. The correctly sequenced recombinant plasmids pGADT7-TaTRX-h, pGADT7-TaPDI, and pGADT7-TaPP2Ac were co-transformed into yeast (Saccharomyces cerevisiae) strain Y2H Gold with pGBKT7-TaNRX1, and then transferred to SD/-Trp-Leu-His-Ade/X-α-gal screening medium. Then on the medium, colonies were found to grow normally and turn blue, which indicated that all the 3 proteins could interact with TaNRX1-D. In order to eliminate false positives, plasmids pCAMBIA1302-CeYFP-TaPDI, pCAMBIA1302-CeYFP-TaPP2Ac, pCAMBIA1302-CeYFP-TaTRX-h and pCAMBIA1302-NeYFP-TaNRX1 were paired, respectively. Then the 3 paired plasmids were co-transformed into Agrobacterium tumefaciens GV3101 and injected into Nicotiana benthamiana leaves. Yellow fluorescent signal was found in the fluorescence confocal microscope, and the yellow signal was mainly located in the nucleus and cell membrane. To verify whether the 3 interacted proteins would respond to drought stress, mannitol was used to simulate drought stress. The recombinant plasmid pYES2-TaPDI, pYES2-TaPP2Ac and pYES2-TaTRX-h and the empty vector pYES2 were transfected into yeast strain BY4741, respectively and cultured in SD/-Ura medium containing 100 mmol/L mannitol, and the growth of the yeasts was observed. The results showed that the growth of yeasts which heterologous expressing TaPDI, TaPP2Ac and TaTRX-h was significantly better than that of empty vector colonies after drought stress, suggesting that TaPDI, TaPP2Ac and TaTRX-h all have positive regulation to mannitol stress. This study validated interaction of 3 candidate proteins with TaNRX1, and simulated drought stress analysis on yeast was conducted, which provide a reference for further elucidating the molecular mechanism of TaNRX1 drought resistance.
|
|
Received: 04 April 2019
|
|
|
|
Corresponding Authors:
zhangxiaoke66@126.com
|
|
|
|
[1] 肖晓琳, 王凯, 张兵,等. 2018. 沟叶结缕草蛋白质二硫键异构酶(ZmPDIs)基因家族耐盐功能分析[J]. 草地学报, 26(05): 142-150.
(Xiao X L, Wang K, Zhang B, et al.2018. Analysis of salt tolerance function of protein disulfide isomerase gene family in Zoysia matrella[J]. Acta Agrestia Sinica, 26(05): 142-150.)
[2] 徐重益. 2007. 小麦抗旱相关基因TaPP2Ac/a的分离、定位与功能验证[D]. 博士论文, 中国农业科学院, 导师: 景蕊莲, pp. 83-93.
(Xu C Y.2007. Isolation, iocalization and functional analysis of drought-resistant gene to TaPP2Ac/a from wheat (Triticum aestivum L.)[D]. Thesis for Ph.D., Chinese Academy of Agricultural Sciences, Supervisor: Jing R L, pp. 83-93.)
[3] 张帆. 2014. 一个普通小麦Trx超家族新基因TaNRX的克隆与抗旱相关标记开发[J]. 作物学报, 40(1): 29-36.
(Zhang F.2014. Cloning a novel gene TaNRX of Trx superfamily and developing its molecular markers related to drought resistance in common wheat[J]. Acta Agronomica Sinica, 40(1): 29-36.)
[4] 张政荣. 2017. 拟南芥蛋白质二硫键异构酶(AtPDIL1)酶活性及其抗逆功能的研究[D]. 硕士学位论文, 山东农业大学, 导师: 王晓云, pp. 55-59.
(Zhang Z R.2017. Enzyme activity and resistance function analysis of protein disulfide isomerase (AtPDIL1) from Arabidopsis[D]. Thesis for M.S., Shandong Agricultural University, Supervisor: Wang X Y, pp. 55-59.)
[5] 赵婷婷. 2016. 硫氧还蛋白调节拟南芥花粉在柱头表面萌发的研究[D]. 硕士学位论文, 山东农业大学, 导师: 高新起; 张宪省, pp. 37-49.
(Zhao T T.2016. Thioredoxin regulates the pollen germination on the stigma in Arabidopsis[D]. Thesis for M.S., Shandong Agricultural University, Supervisor: Gao X Q; Zhang X S , pp. 37-49.)
[6] Funato Y, Michiue T, Asashima M, et al.2006. The thioredoxin-related redox-regulating protein nucleoredoxin inhibits Wnt-β-catenin signalling through dishevelled[J]. Nature Cell Biology, 8(5): 501-508.
[7] Funato Y, Miki H.2007. Nucleoredoxin, a novel thioredoxin family member involved in cell growth and differentiation[J]. Antioxidants & Redox Signalling, 9(8): 1035-1057.
[8] Jaime A R, Karina R G, Joseph I, et al.2018. Ethanol targets nucleoredoxin/dishevelled interactions and stimulates phosphatidylinositol 4-phosphate production in vivo and in vitro[J]. Biochemical Pharmacology, 156: 135-146.
[9] Kaufman R J.1999. Stress signaling from the lumen of the endoplasmic reticulum: Coordination of gene transcriptional and translational controls[J]. Genes & Development, 13(10): 1211-1233.
[10] Kneeshaw S, Keyani R, Delorme-Hinoux V, et al.2017. Nucleoredoxin guards against oxidative stress by protecting antioxidant enzymes[J]. Proceedings of the National Academy of Sciences of the USA, 114(31): 8414-8419.
[11] Kurooka H, Kato K, Minoguchi S, et al.1997. Cloning and characterization of the nucleoredoxin gene that encodes a novel nuclear protein related to thioredoxin[J]. Genomics, 39(3): 331-339.
[12] Laughner B J, Ferl S R J.1998. A novel nuclear member of the thioredoxin superfamily[J]. Plant Physiology, 118(3): 987-996.
[13] Lechward K, Sugajska E, Baere I D, et al.2006. Interaction of nucleoredoxin with protein phosphatase 2A[J]. FEBS Letters, 580(15): 3631-3637.
[14] Marchal C, Delorme-Hinoux V, Bariat L, et al.2014. NTR/NRX define a new thioredoxin system in the nucleus of Arabidopsis thaliana cells[J]. Molecular Plant, 7(1): 30-44.
[15] Nakao L S, Everley R A, Marino S M, et al.2015. Mechanism-based proteomic screening identifies targets of thioredoxin-like proteins[J]. Journal of Biological Chemistry, 290(9): 5685-5695.
[16] Pulido P, Cazalis R, Cejudo F J.2011. An antioxidant redox system in the nucleus of wheat seed cells suffering oxidative stress[J]. Plant Journal, 57(5): 132-145.
[17] Qin Y, Leydon A R, Manziello A, et al.2009. Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil[J]. PLOS Genetics, 5(8): 1-19.
[18] Serrato A J, Cejudo F J.2003. Type-h thioredoxins accumulate in the nucleus of developing wheat seed tissues suffering oxidative stress[J]. Planta, 217(3): 392-399.
[19] Traverso J A, Florence V, Roland C, et al.2007. PsTRXh1 and PsTRXh2 are both pea h-type thioredoxins with antagonistic behavior in redox imbalances[J]. Plant Physiology, 143(1): 300-311.
[20] Urbainsky C,Nölker R, Imber M, et al.2018. Nucleoredoxin-dependent targets and processes in neuronal cells[J]. Oxidative Medicine and Cellular Longevity, 2018: 4829872.
[21] Vazquez-Tello A, Ouellet F, Sarhan F.1998. Low temperature-stimulated phosphorylation regulates the binding of nuclear factors to the promoter of Wcs120, a cold-specific gene in wheat[J]. Molecular & General Genetics, 257(2): 157-166.
[22] Zhang T, Zhu M, Zhu N, et al.2016. Identification of thioredoxin targets in guard cell enriched epidermal peels using cysTMT proteomics[J]. Journal of Proteomics, 133: 48-53. |
|
|
|
