苹果MdWRKY18和MdWRKY40参与盐胁迫途径分子机理研究

doi:10.3864/j.issn.0578-1752.2018.23.010

摘要/Abstract

摘要：

【目的】研究苹果WRKY转录因子MdWRKY18和MdWRKY40蛋白结构、表达水平及在盐胁迫中的功能,为进一步完善盐胁迫分子机理的研究提供参考。【方法】以‘红脆2号’苹果为试材,克隆MdWRKY18和MdWRKY40,对其蛋白结构进行分析;采用qRT-PCR测定该基因在盐胁迫条件下的表达水平,并通过GUS染色分析它们启动子活性,利用酵母双杂分析其互作关系,并通过转基因验证其功能。【结果】蛋白结构分析表明MdWRKY18和MdWRKY40均含有一个WRKY、Cx5C以及HxH结构域;MdWRKY18和MdWRKY40表达水平和启动子活性受150 mmol·L ^-1 NaCl诱导,酵母双杂交试验表明,MdWRKY18和MdWRKY40能够和自身互作形成同源二聚体,且MdWRKY18也能和MdWRKY40互作形成异源二聚体;在‘王林’愈伤中分别过表达MdWRKY18和MdWRKY40时,能够促进MdSOS1和MdNHX1的表达,并提高‘王林’愈伤在盐胁迫处理下的生长量,在‘王林’愈伤中共表达MdWRKY18和MdWRKY40时,同样能够促进MdSOS1和MdNHX1的表达,但对提高‘王林’愈伤的生长量要高于分别过表达MdWRKY18和MdWRKY40。【结论】苹果MdWRKY18和MdWRKY40受盐胁迫的诱导,可以形成同源或异源二聚体,并增强‘王林’愈伤对盐胁迫的耐性。

关键词: 苹果, WRKY转录因子, 盐胁迫, GUS染色, 酵母双杂交

Abstract:

【Objective】 In order to improve the molecular mechanism of salt stress, we studied several aspects of MdWRKY18 and MdWRKY40 in apple WRKY transcription factors, including the protein structure, the expression level and the function in salt stress. 【Method】 We cloned the MdWRKY18 and MdWRKY40 genes in ‘Hongcui No.2’ apple and analysed their protein structure. The expression levels of MdWRKY18 and MdWRKY40 were studied by the qRT-PCR under the salt stress, and their promoter activities were analyzed using the GUS staining. We analyzed the interaction relationship between MdWRKY18 and MdWRKY40 proteins by yeast two-hybrid and verified their function by transgenosis. 【Result】 Analysis of protein structure showed that both MdWRKY18 and MdWRKY40 proteins contained a WRKY, Cx5C and HxH structural domains. The expression levels and promoter activities of MdWRKY18 and MdWRKY40 were induced by the 150 mmol·L ^-1 NaCl. The yeast two-hybrid experiments showed that MdWRKY18 and MdWRKY40 could respectively interact with itself to form homodimers, and MdWRKY18 could also interact with MdWRKY40 to form heterodimers. When MdWRKY18 and MdWRKY40 was overexpressed respectively in orin callus, they could increase the callus weight under salt stress and promote the expression of MdSOS1 and MdNHX1. When MdWRKY18 and MdWRKY40 were co-overexpressed in orin callus, it could also promote the expression of MdSOS1 and MdNHX1, however, the weight of callus was heavier than the weight of callus overexpressing MdWRKY18 or MdWRKY40. 【Conclusion】 MdWRKY18 and MdWRKY40 were induced by the salt stress, and they could form homodimers or heterodimers, overexpressing MdWRKY18 or MdWRKY40 in orin callus could increase its salt tolerance.

Key words: apple, WRKY transcription factor, salt stress, GUS staining, yeast two-hybrid

许海峰,杨官显,张静,邹琦,王意程,曲常志,姜生辉,王楠,陈学森. 苹果MdWRKY18和MdWRKY40参与盐胁迫途径分子机理研究[J]. 中国农业科学, 2018, 51(23): 4514-4521.

XU HaiFeng,YANG GuanXian,ZHANG Jing,ZOU Qi,WANG YiCheng,QU ChangZhi,JIANG ShengHui,WANG Nan,CHEN XueSen. Molecular Mechanism of Apple MdWRKY18 and MdWRKY40 Participating in Salt Stress[J]. Scientia Agricultura Sinica, 2018, 51(23): 4514-4521.

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参考文献 42

[1]	MAHAJAN S, TUTEJA N . Cold, salinity and drought stresses: An overview. Archives of Biochemistry and Biophysics, 2005,444:139-158. doi: 10.1016/j.abb.2005.10.018 pmid: 16309626
[2]	TUTEJA N . Mechanisms of high salinity tolerance in plants. Methods in Enzymology, 2007,428:419-438. doi: 10.1016/S0076-6879(07)28024-3
[3]	DONG D, ZHANG L F, HANG W, LIU Z J, ZHANG Z X, ZHENG Y L . Differential expression of miRNAs in response to salt stress in maize roots. Annals of Botany, 2009,103:29-38. doi: 10.1093/aob/mcn205 pmid: 18952624
[4]	OHTA M, HAYASHI Y, NAKASHIMA A, HAMADA A, TANAKA A, NAKAMURA T, HAYAKAWA T . Introduction of a Na ⁺/H ⁺ antiporter gene from Atriplex gmelini confers salt tolerance to rice. FEBS Letters, 2002,532:279-282.
[5]	NING Z, CHENG S, LIU X, HAO D, DAI M, ZHOU D X, YANG W, YU Z . The R2R3-type MYB gene OsMYB91 has a function in coordinating plant growth and salt stress tolerance in rice. Plant Science, 2015,236:146-156. doi: 10.1016/j.plantsci.2015.03.023 pmid: 26025528
[6]	XUE Z Y, ZHI D Y, XUE G P, ZHANG H, ZHAO Y X, XIA G M . Enhanced salt tolerance of transgenic wheat (Tritivum aestivum L.) expressing a vacuolar Na ⁺/H ⁺ antiporter gene with improved grain yields in saline soils in the field and a reduced level of leaf Na ⁺. Plant Science , 2004,167:849-859. doi: 10.1016/j.plantsci.2004.05.034
[7]	ÇIÇEK N, HÇ X . Effects of salt stress on some physiological and photosynthetic parameters at three different temperatures in six soya bean (Glycine max L. Merr.) Cultivars. Journal of Agronomy and Crop Science, 2008,194:34-46. doi: 10.1111/j.1439-037X.2007.00288.x
[8]	HUSAINI A M, ABDIN M Z . Development of transgenic strawberry (Fragaria × ananassa Duch.) plants tolerant to salt stress. Plant Science, 2008,174:446-455.
[9]	RASHEDY A . Response of two grape rootstocks to some salt tolerance treatments under saline water conditions. Journal of Horticultural Science & Ornamental Plants, 2010,2:93-106.
[10]	XUE H, ZHANG F, ZHANG Z H, FU JF, WANG F, ZHANG B, MA Y . Differences in salt tolerance between diploid and autotetraploid apple seedlings exposed to salt stress. Scientia Horticulturae, 2015,190:2430. doi: 10.1016/j.scienta.2015.04.009
[11]	SAHI C, SINGH A, BLUMWALD E, GROVER A . Beyond osmolytes and transporters: Novel plant salt-stress tolerance-related genes from transcriptional profiling data. Physiologia Plantarum, 2006,127:1-9. doi: 10.1111/j.1399-3054.2005.00610.x
[12]	HASEGAWA P M, BRESSAN R A, ZHU J K, BOHNERT H J . Plant cellular and molecular responses to high salinity. Annual Review of Plant Biology, 2000,51:463-499. doi: 10.1146/annurev.arplant.51.1.463
[13]	BLUMWALD E, POOLE R J . Na/H antiport in isolated tonoplast vesicles from storage tissue of beta vulgaris. Plant Physiology, 1985,78:163-167. doi: 10.1104/pp.78.1.163 pmid: 16664191
[14]	DING Z, LI S, AN X, LIU X, QIN H, WANG D . Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana. Journal of Genetics and Genomics, 2009,36:17-29. doi: 10.1016/S1673-8527(09)60003-5 pmid: 19161942
[15]	ABE H, URAO T, ITO T, SEKI M, SHINOZAKI K, YAMAGUCHISHINOZAKI K . Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. The Plant Cell, 2003,15:63-78. doi: 10.1105/tpc.006130 pmid: 12509522
[16]	YANG O, POPOVA O V, SÜTHOFF U, LÜKING I, DIETZ K J, GOLLDACK D . The Arabidopsis basic leucine zipper transcription factor AtbZIP24 regulates complex transcriptional networks involved in abiotic stress resistance. Gene, 2009,436:45-55. doi: 10.1016/j.gene.2009.02.010 pmid: 19248824
[17]	REN X, CHEN Z, LIU Y, ZHANG H, ZHANG M, LIU Q, HONG X, ZHU J K, GONG Z . ABO3, a WRKY transcription factor, mediates plant responses to abscisic acid and drought tolerance in Arabidopsis. The Plant Journal, 2010,63:417-429. doi: 10.1111/j.1365-313X.2010.04248.x pmid: 20487379
[18]	JIANG Y, YANG B, DEYHOLOS M K . Functional characterization of the Arabidopsis bHLH92 transcription factor in abiotic stress. Molecular Genetics and Genomics, 2009,282:503-516.
[19]	WU X, SHIROTO Y, KISHITANI S, ITO Y, TORIYAMA K . Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter. Plant Cell Reports, 2009,28:21-30. doi: 10.1007/s00299-008-0614-x pmid: 2018818929
[20]	JIANG Y J, LIANG G, YU D Q . Activated expression of WRKY57 confers drought tolerance inArabidopsis. Molecular Plant, 2012,5:1375-1388. doi: 10.1093/mp/sss080 pmid: 22930734
[21]	JIANG Y, DEYHOLOS M . Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Molecular Biology, 2009,69:91-105.
[22]	XU H F, WANG N, LIU J X, QU C Z, WANG Y C, JIANG S H, LU N L, WANG D Y, ZHANG Z Y, CHEN X S . The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes. Plant Molecular Biology, 2017,94:149-165. doi: 10.1007/s11103-017-0601-0 pmid: 28286910
[23]	KENNETH J L, THOMAS D S . Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^-△△CT method . Methods, 2001,25:402-408. doi: 10.1006/meth.2001.1262
[24]	ZHANG Y, WANG L . The WRKY transcription factor superfamily: Its origin in eukaryotes and expansion in plants. BMC Evolutionary Biology, 2005,5(1):1-12. doi: 10.1186/1471-2148-5-1 pmid: 15629062
[25]	RUSHTON P J, SOMSSICH I E, RINGLER P, SHEN Q J . WRKY transcription factors. Trends in Plant Science, 2010,15:247-258. doi: 10.1016/j.tplants.2010.02.006
[26]	WU K L, GUO Z J, WANG H H, LI J . The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Research, 2005,12:9-26.
[27]	GUO D, ZHANG J, WANG X, HAN X, WEI B, WANG J . The WRKY transcription factor WRKY71/EXB1 controls shoot branching by transcriptionally regulating RAX genes in Arabidopsis. The Plant Cell, 2015,27:3112-3127. doi: 10.1105/tpc.15.00829 pmid: 26578700
[28]	SCHLUTTENHOFER C, YUAN L . Regulation of specialized metabolism by WRKY transcription factors. Plant Physiology, 2015,167:295-306. doi: 10.1104/pp.114.251769 pmid: 25501946
[29]	ZHANG L, GU L, RINGLER P, SMITH S, RUSHTON P J, SHEN Q J . Three WRKY transcription factors additively repress abscisic acid and gibberellins signaling in aleurone cells. Plant Science, 2015,236:214-222. doi: 10.1016/j.plantsci.2015.04.014 pmid: 26025535
[30]	CHEN L, SONG Y, LI S, ZHANG L, ZOU C, YU D . The role of WRKY transcription factors in plant abiotic stresses. Biochimica et Biophysica Acta, 2012,1819:120-128. doi: 10.1016/j.bbagrm.2011.09.002 pmid: 21964328
[31]	BANERJEE A, ROYCHOUDHURY A . WRKY proteins: signaling and regulation of expression during abiotic stress responses. The Scientific World Journal, 2015,2015:3-24. doi: 10.1155/2015/807560 pmid: 4387944
[32]	ZHOU Q Y, TIAN A G, ZOU H F, XIE Z M, LEI G, HUANG J, WANG C M, WANG H W, ZHANG J S, CHEN SY . Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnology Journal, 2008,6:486-503. doi: 10.1111/j.1467-7652.2008.00336.x pmid: 18384508
[33]	JIANG Y, DEYHOLOS M K . Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes. BMC Plant Biology, 2006,6:25. doi: 10.1186/1471-2229-6-25 pmid: 1621065
[34]	BERRI S, ABBRUSCATO P, FAIVRE-RAMPANT O, BRASILEIRO A C, FUMASONI I, SATOH K, KIKUCHI S, MIZZI L, MORANDINI P, PÈ M E, PIFFANELLI P . Characterization of WRKY co-regulatory networks in rice and Arabidopsis. BMC Plant Biology, 2009,9:120. doi: 10.1186/1471-2229-9-120 pmid: 19772648
[35]	NIU C F, WEI W, ZHOU Q Y, TIAN A G, HAO Y J, ZHANG W K, MA B, LIN Q, ZHANG Z B, ZHANG J S, CHEN S Y . Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants. Plant Cell and Environment, 2012,35:1156-1170. doi: 10.1111/j.1365-3040.2012.02480.x pmid: 22220579
[36]	QIN Y, TIAN Y, LIU X . A wheat salinity-induced WRKY transcription factor TaWRKY93 confers multiple abiotic stress tolerance in Arabidopsis thaliana. Biochemical And Biophysical Research Communications, 2015,464:428-433. doi: 10.1016/j.bbrc.2015.06.128 pmid: 26106823
[37]	ZHOU L, WANG N N, GONG S Y, LU R, LI Y, LI X B . Overexpression of a cotton (Gossypium hirsutum) WRKY gene, GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants. Plant Physiology and Biochemistry, 2015,96:311-320.
[38]	CHEN H, LAI Z B, SHI J W, XIAO Y, CHEN Z X, XU X P . Roles of Arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biology, 2010,10:281. doi: 10.1186/1471-2229-10-281 pmid: 21167067
[39]	WANG N, QU C Z, WANG Y C, XU H F, JIANG S H, FANG H C, LIU J X, ZHANG Z Y, CHEN X S . MdMYB4 enhances apple callus salt tolerance by increasing MdNHX1 expression levels. Plant Cell, Tissue and Organ Culture, 2017,131:283-293. doi: 10.1007/s11240-017-1283-7
[40]	XU X, CHEN C, FAN B, CHEN Z . Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors. The Plant Cell, 2006,18:1310-1326. doi: 10.1105/tpc.105.037523 pmid: 16603654
[41]	CHEN Y F, LI L Q, XU Q, KONG Y H, WANG H, WU W H . The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis. The Plant Cell, 2009,21:3554-3566. doi: 10.1105/tpc.108.064980 pmid: 19934380
[42]	BESSEAU S, LI J, PALVA E T . WRKY54 and WRKY70 cooperate as negative regulators of leaf senescence in Arabidopsis thaliana. Journal of Experimental Botany, 2012,63:2667-2679.