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 FENG Qian,CHEN Yongfu,YAO Yinan,et al.Response of heterologous overexpression of Populus euphratica PeGRF6/8a in tobacco under different stresses[J].Chinese Journal of Applied & Environmental Biology,2019,25(03):665-671.[doi:10.19675/j.cnki.1006-687x.201810006]





Response of heterologous overexpression of Populus euphratica PeGRF6/8a in tobacco under different stresses
西南科技大学生命科学与工程学院 绵阳 621010
FENG Qian CHEN Yongfu YAO Yin’an WU Yingqing ZHANG Guoyang HAN Ying & GAO Yongfeng**
School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China
PeGRF6/8a 14-3-3 protein abscisic acid tobacco low nitrogen high nitrogen salt stress
Q945.78 : Q78
14-3-3蛋白是一种广泛存在于动植物体内的高度保守的蛋白家族,通过与各种靶蛋白之间的互作,参与生物体内各种生理生化过程和代谢反应,且在生物与非生物胁迫响应中发挥着重要作用. 为明确胡杨PeGRF6/8a在非生物胁迫中的功能,克隆了胡杨14-3-3蛋白家族中PeGRF6/8a的cDNA序列,构建该基因的植物过表达载体pBI121-35S::PeGRF6/8a,并采用农杆菌介导法将其转化野生型烟草. 对获得的异源过表达烟草进行以下处理:(1)浓度为1 μmol/L和2.5 μmol/L脱落酸(Abscisic acid,ABA)处理条件下的种子萌发实验;(2)低氮(2.5 mmol/L KNO3)和高氮(150 mmol/L KNO3)胁迫下的根长对比实验;(3)1/2霍格兰(Hongland)营养液水培实验,分别设置对照(CK)、低氮(0.2 mmol/L KNO3)、高氮(150 mmol/L KNO3)和盐胁迫(150 mmol/L NaCl)4个处理. 结果显示:(1)在ABA处理下,转基因烟草种子的萌发率较野生型低,且在2.5 μmol/L处理下更加显著;(2)低氮处理下,转基因烟草的根长显著短于野生型烟草的根长,而高氮处理下的结果相反;(3)水培实验中,低氮和盐胁迫处理下,转基因烟草与野生型相比,其下部的叶片明显变黄或萎焉,所含叶绿素和类胡萝卜素含量明显降低,抗氧化酶SOD以及渗透调节物质脯氨酸和可溶性蛋白的含量也显著降低,而叶片中丙二醛(MDA)的积累量却显著升高;高氮胁迫下,野生型烟草的萎蔫速度要显著快于转基因烟草,其转基因植株的生理指标除MDA积累量外均显著高于野生型. 上述结果表明在烟草中异源过表达胡杨PeGRF6/8a能够降低植物对低氮和盐的耐受性,但可以增强植物对高氮的耐受性. (图9 参31
14-3-3 Protein is a type of highly conserved protein family widely distributed both in animals and plants. It is not only involved in various physiological and biochemical processes and metabolic reactions in organisms by interacting with different target proteins but also plays important roles in response to biotic and abiotic stresses. To clarify the function of the PeGRF6/8a under abiotic stress, we cloned the PeGRF6/8a from the 14-3-3 protein family of Populus euphratica, constructed pBI121-35S::PeGRF6/8a, an expression vector, and then transformed it into wild-type tobacco by Agrobacterium-mediated transformation. The obtained transgenic tobaccos were treated as follows: (1) Seed germination experiments under 1 μmol/L and 2.5 μmol/L abscisic acid (ABA) treatments; (2) Root length comparison experiments under low nitrogen (2.5 mmol/L KNO3) and high nitrogen (150 mmol/L KNO3) treatments; (3) 1/2 Hoagland hydroponics experiments—four treatments that included control (CK), low nitrogen (0.2 mmol/L KNO3), high nitrogen (150 mmol/L KNO3), and salt stress (150 mmol/L NaCl). The results were as follows: (1) Under ABA treatment, the germination rate of transgenic tobacco seeds was lower than that in the wild type. Upon increased ABA concentration, the germination rate of transgenic tobacco seeds declined much faster than that of the wild type; (2) Under low nitrogen treatment, the root length of transgenic tobacco was distinctly shorter than that of wild type tobacco. However, it was in contrast with the root length under high nitrogen treatment. (3) Under low nitrogen and salt treatments, compared to the wild type, in transgenic tobacco, the lower leaves showed yellowing or wilting and the chlorophyll and carotenoid content in the leaves were markedly lower. Furthermore, the activity of superoxide dismutase and osmolyte content such as proline and soluble proteins was significantly lower in transgenic than in wild-type plants but the malondialdehyde (MDA) content was higher. However, under high nitrogen treatment, the speed of plants wilting in the wild type was markedly faster than that in the transgenic plants, and the resistance and related physiological indexes (except MDA) of transgenic plants were considerably higher than those of wild type. These results indicate that heterologous overexpression of P. euphratica PeGRF6/8a in tobacco can weaken plant tolerance to low nitrogen and salt stresses, but it can enhance plant tolerance under high nitrogen stress.


1. Boston PF, Jackson P, Kynoch PA, Thompson RJ. Purification, properties, and immunohistochemical localisation of human brain 14-3-3 protein [J]. J Neurochem, 1982, 38 (5): 1466-1474
2. Fu H, Subramanian RR, Master SC. 14-3-3 Proteins: structure, function, and regulation [J]. Annu Rev Pharmacol Toxicol, 2000, 40 (1): 617-647
3. Aitken A. 14-3-3 Proteins: a historic overview [J]. Semin Cancer Biol, 2006, 16 (3): 162-172
4. Aitken A, Howell S, Jones D, Madrazo J, Martin H, Patel Y, Robinson K. Post-translationally modified 14-3-3 isoforms and inhibition of protein kinase C [J]. Mol Cell Biochem. 1995, 149 (150): 41-49
5. Denison FC, Paul AL, Zupanska AK, Ferl RJ. 14-3-3 Proteins in plant physiology [J]. Semin Cell Dev Biol, 2011, 22 (7): 720-727
6. Camoni L, Visconti S, Aducci P, Marra M. 14-3-3 Proteins in plant hormone signaling: doing several things at once [J]. Front Plant Sci, 2018, 9: 297
7. Del VF, Casaretto JA, Quatrano RS. 14-3-3 Proteins are components of the transcription complex of ATEM1 promoter in Arabidopsis [J]. Planta, 2007, 227 (1): 167-175
8. Chen YX, Zhou XJ, Chang S, Chu ZL, Wang HM, Han SC, Wang YD. Calcium-dependent protein kinase 21 phosphorylates 14-3-3 proteins in response to ABA signaling and salt stress in rice [J]. Biochem Biophys Res Commun, 2017, 493 (4): 1450-1456
9. Shin R, Alvarez S, Burch AY, Jez JM, Schachtman DP. Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes [J]. PNAS, 2007, 104 (15): 6460-6465
10. Shin R, Jez JM, Basra A, Zhang B, Schachtman DP. 14-3-3 Proteins fine-tune plant nutrient metabolism [J]. FEBS Lett, 2011, 585 (1): 143-147
11. Zhang Y, Zhao H, Zhou SY, He Y, Luo QC, Zhang F, Qiu D, Feng JL, Wei QH, Chen LH, Chen MJ, Chang JL, Yang GX, He GY. Expression of TaGF14b, a 14-3-3 adaptor protein gene from wheat, enhances drought and salt tolerance in transgenic tobacco [J]. Planta, 2018, 248 (1): 117-137
12. Zhou H, Lin H, Chen S,Becker K, Yang Y, Zhao J, Kudla J, Schumaker KS, Guo Y. Inhibition of the Arabidopsis salt overly sensitive pathway by 14-3-3 proteins [J]. Plant Cell, 2014, 26 (3): 1166–1182
13. Zhang ZT, Zhou Y, Li Y, Shao SQ, Li BY, Shi HY, Li XB. Interactome analysis of the six cotton 14-3-3s that are preferentially expressed in fibres and involved in cell elongation [J]. J Exp Bot, 2010, 61 (12): 3331–3344
14. Chang IF, Curran A, Woolsey R, Quilici D, Cushman JC, Mittler R, Harmon A, Harper JF. Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana [J]. Proteomics, 2009, 9 (11): 2967-2985
15. Tian F, Wang T, Xie Y, Zhang J, Hu J. Genome-wide identification, classification, and expression analysis of 14-3-3 gene family in Populus [J]. PloS One, 2015, 10 (4): e0123225
16. 陈永富, 王阳, 冯倩, 唐海, 吴英青, 高永峰. 异源表达胡杨PeCPD基因提高烟草对盐、高氮和干旱胁迫的抗性[J]. 应用与环境生物学报, 2017, 23 (2): 225-231 [Chen YF, Wang Y, Feng Q, Tang H, Wu YQ, Gao YF. Heterlogous overexpression of Populus Euphratica CPD improved tobacco resistance to salt, high nitrogen, and drought stress [J]. Chin J Appl Environ Biol, 2017, 23 (2): 225-231]
17. 邹琦. 植物生理学[M]. 北京: 中国农业出版社, 2000: 163-166 [Zou Q. Plant Physiology [M]. Beijing: China Agriculture Press, 2000: 163-166]
18. Lichtenthaler HK, Wellburn AR. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents [J]. Anal Peach, 1983, 11 (5): 591-592
19. 贾中民, 魏虹, 孙晓灿, 李昌晓, 孟飞翔, 谢小红. 秋华柳和枫杨幼苗对镉的积累和耐受性[J]. 生态学报, 2011, 31 (1): 107-114 [Jia ZM, Wei H, Sun XC, Li CX, Meng FX, Xie XH. Accumulation and tolerance of Salix varegate and Pterocarya stenoptera seedlings to cadmium [J]. Chin J Ecol, 2011, 31 (1): 107-114]
20. Read SM, Northcote DH. Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein [J]. Anal Biochem, 1981, 116 (1): 53-64
21. 高俊凤. 植物生理学实验指导[M]. 北京: 高等教育出版社, 2006: 210 [Gao JF. Plant Physiology Experimental Guide [M]. Beijing: Higher Education Press, 2006: 210]
22. 唐海. 胡杨中液泡膜水通道蛋白PeTIP1;2基因功能及其启动子研究[D]. 绵阳: 西南科技大学, 2018 [Tang H. Function studies on PeTIP1;2 gene and its promoter effects of tonoplast aquaporin in Populus euphratica [D]. MianYang: Southwest University of Science and Technology, 2018]
23. Booij PP, Roberts MR, Vogelzang SA, Kraayenhof R, Boer DAH. 14-3-3 proteins double the number of outward-rectifying K+ channels available for activation in tamato cells [J]. Plant J, 1999, 20 (6): 673683
24. Wijngaard PW, Sinnige MP, Roobeek I, Reumer A, Schoonheim PJ, Mol JN, Wang M, Boer DAH. Abscisic acid and 14-3-3 proteins control K+ channel activity in barley embryonic root [J]. Plant J, 2005, 41 (1): 43-55
25. Balotf S, Kavoosi G, Kholdebarin B. Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedings [J]. Biotechnol Appl Biochem, 2016, 63 (2): 220-229
26. Lambeck I, Chi JC, Krizowski S, Mueller S, Mehlmer N, Teige M, Fischer K, Schwarz G. Kinetic analysis of 14-3-3-inhibited Arabidopsis thaliana nitrate reductase [C]. Biochemistry, 2010, 49 (37): 8177-8186
27. Xu HN, Zhao XL, Guo CL, Chen LM, Li KZ. Spinach 14-3-3 protein interacts with the plasma membrane H(+)-ATPase and nitrate reductase in response to excess nitrate stress [J]. Plant Physiol Biochem, 2016, 106: 187-197
28. Waqas M, Feng SZ, Amjad H, Letuma P, Zhan WS, Zhong L, Fang CG, Arafat Y, Khan MU, Tayyab M, Lin WX. Protein Phosphatase (PP2C9) induces protein expression differentially to mediate nitrogen utilization efficiency in rice under Nitrogen-Deficient condition [J]. Int J Mol Sci, 2018, 19 (9)
29. 赵秀玲. 14-3-3蛋白在菠菜中的克隆及应答高NO3-的作用机理研究[D]. 云南: 昆明理工大学, 2013 [Zhao XL. Cloning and mechanism study of spinach 14-3-3 protein (So14-3-3) in response to high NO3- nitrate stress [D]. YunNan: Kunming University of Science and Technology, 2013]
30. Zhou HP, Lin HX, Chen S, Becker K, Yang Y, Zhao J, Kudla J, Schumaker KS, Guo Y. Inhibition of the Arabidopsis salt overly sensitive pathway by 14-3-3 proteins [J]. Plant Cell, 2014, 26 (3): 1166-1182
31. 肖强,郑海雷. 14-3-3蛋白与植物细胞信号转导[J]. 中国细胞生物学学报, 2005, 27 (4):417-422 [Xiao Q, Zheng HL. 14-3-3 protein and plant cell signal transduction [J]. Chin Cell Biol, 2005, 27 (4): 417-422]

更新日期/Last Update: 2019-06-25