逆境诱导基因GsSNARE1的耐逆功能分析

doi:10.3864/j.issn.0578-1752.2013.11.002

摘要/Abstract

摘要： 【目的】分离GsSNARE1，并分析其耐逆功能，为深入研究GsSNARE1功能及分子机制奠定基础。【方法】以耐盐野生大豆G50109为材料，利用酵母双杂交验证GsSNARE1与GsCBRLK的互作关系，通过real-time PCR分析干旱和盐处理后GsSNARE1的表达特性，对GsSNARE1进行原核表达，分析GsSNARE1抗盐旱功能。【结果】获得与GsCBRLK互作的GsSNARE1，同源克隆其全长CDS，并在酵母体内验证了二者的相互作用；Real-time PCR分析表明GsSNARE1受盐、干旱胁迫诱导，经PLACE分析，发现其启动子中含有多个逆境胁迫相关的顺式作用元件；GsSNARE1在植物不同组织中均表达；GsSNARE1的表达降低了重组菌对盐、干旱胁迫的耐性。【结论】验证了GsSNARE1与GsCBRLK的互作关系，GsSNARE1在不同组织中均表达，且受盐、干旱胁迫诱导，GsSNARE1的表达降低了重组菌耐盐、耐旱能力。

关键词: 野生大豆 , SNARE蛋白 , 功能分析 , 盐胁迫 , 干旱胁迫

Abstract: 【Objective】The aim of this study is to isolate GsSNARE1, characterize its function under environmental stress, and provide an important basis for studying the precise function and molecular mechanism of GsSNARE1.【Method】Glycine soja 50109 was used as gene cloning material, and the interaction between GsSNARE1 and GsCBRLK was verified by yeast two hybrid analysis. Real-time PCR analysis was used to analyze the expression profile of GsSNARE1 under stress conditions and in different plant tissues. The GsSNARE1 protein was expressed in E. coli, and its function was analyzed under salt and drought stresses.【Result】In this study, the GsCBRLK interacting protein GsSNARE1 was isolated, the full length GsSNARE1 gene was cloned, and the interaction between GsSNARE1 and GsCBRLK in yeast NMY51 was verified. Real-time PCR analysis showed that expression of GsSNARE1 was greatly induced by salt and drought stresses, and PLACE analysis revealed a serial of stress-related cis-elements in GsSNARE1 promoter. GsSNARE1 expressed in different tissues of G. soja. GsSNARE1 expression decreased salt and drought resistance of the recombinant E.coli.【Conclusion】GsSNARE1 interacted with GsCBRLK in yeast. GsSNARE1 transcripts were greatly accumulated under salt and drought stresses, and were detected in different tissues. Expression of GsSNARE1 in E.coli resulted in decreased salt and drought tolerance.

Key words: Glycine soja , SNARE protein , functional characterization , salt stress , drought stress

孙明哲, 孙晓丽, 于清悦, 纪巍, 才华, 朱延明. 逆境诱导基因GsSNARE1的耐逆功能分析[J]. 中国农业科学, 2013, 46(11): 2191-2200.

SUN Ming-Zhe, SUN Xiao-Li, YU Qing-Yue, JI Wei, CAI Hua, ZHU Yan-Ming. Functional Analysis of a Stress-Induced SNARE Gene GsSNARE1 in Response to Salt and Drought Stresses[J]. Scientia Agricultura Sinica, 2013, 46(11): 2191-2200.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2013.11.002

https://www.chinaagrisci.com/CN/Y2013/V46/I11/2191

参考文献

[1]Dornbos D L, Jr, Mullen R E. Soybean seed protein and oil contents and fatty acid composition adjustments by drought and temperature. Journal of the American Oil Chemists Society, 1992, 69(3): 228-231.

[2]Ashraf M. Breeding for salinity tolerance in plants. Critical Reviews in Plant Sciences, 1994, 13(1): 17-42.

[3]Subramanyam K, Arun M, Mariashibu T S, Theboral J, Rajesh M, Singh N K, Manickavasagam M, Ganapathi A. Overexpression of tobacco osmotin (Tbosm) in soybean conferred resistance to salinity stress and fungal infections. Planta, 2012, 236(6): 1909-1925.

[4]Bihmidine S, Lin JS, Stone J M, Awada T, Specht J E, Clemente T E. Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress. Planta, 2013, 237(1): 55-64.

[5]Liu M, Li D, Wang Z, Meng F, Li Y, Wu X, Teng W, Han Y, Li W. Transgenic expression of ThIPK2 gene in soybean improves stress tolerance, oleic acid content and seed size. Plant Cell, Tissue and Organ Culture, 2012, 111(3): 277-289.

[6]Seo J S, Sohn H B, Noh K, Jung C, An J H, Donovan C M, Somers D A, Kim D I, Jeong S C, Kim C G, Kim H M, Lee S H, Choi Y D, Moon T W, Kim C H, Cheong J J. Expression of the Arabidopsis AtMYB44 gene confers drought/salt-stress tolerance in transgenic soybean. Molecular Breeding, 2012, 29: 601-608.

[7]Xue R G, Zhang B, Xie H F. Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress. Plant Cell, Tissue and Organ Culture, 2007, 89: 177-183.

[8]Sutter J U, Campanoni P, Tyrrell M, Blatt M R. Selective mobility and sensitivity to SNAREs is exhibited by the Arabidopsis KAT1 K+ channel at the plasma membrane. The Plant Cell, 2006, 18: 935-954.

[9]Kalde M, Nuhse T S, Findlay K, Peck S C. The syntaxin SYP132 contributes to plant resistance against bacteria and secretion of pathogenesis-related protein 1. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(28): 11850-11855.

[10]Bassham D C, Blatt M R. SNAREs: Cogs and coordinators in signaling and development. Plant Physiology, 2008, 147: 1504-1515.

[11]Sokolovski S, Hills A, Gay R A, Blatt M R. Functional interaction of the SNARE protein NtSyp121 in Ca2+ channel gating, Ca2+ transients and ABA signalling of stomatal guard cells. Molecular Plant, 2008, 1(2): 347-358.

[12]Yano D, Sato M, Saito C, Sato M H, Morita M T, Tasaka M. A SNARE complex containing SGR3/AtVAM3 and ZIG/VTI11 in gravity-sensing cells is important for Arabidopsis shoot gravitropism. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(14): 8589-8594.

[13]Uemura T, Ueda T, Ohniwa R L, Nakano A, Takeyasu K, Sato M H. Systematic analysis of SANRE molecules in Arabidopsis: Dissection of the post-Golgi network in plant cells. Cell Structure and Function, 2004, 29: 49-65.

[14]金红敏, 李立新. 拟南芥SNARE因子在膜泡运输中的功能. 植物学报, 2010, 45(4): 479-491.

Jin H M, Li L X. Role of Arabidopsis SNARE proteins in vesicle trafficking. Chinese Bulletin of Botany, 2010, 45(4): 479-491. (in Chinese)

[15]Leyman B, Geelen D, Quintero F J, Blatt M R. A tobacco syntaxin with a role in hormonal control of guard cell ion channels. Science, 1999, 283: 537-540.

[16]Leyman B, Geelen D, Blatt M R. Localization and control of expression of NtSyr1, a tobacco SNARE protein. The Plant Journal, 2000, 24(3): 369-381.

[17]Zhu J, Gong Z, Zhang C, Song C P, Damsz B, Inan G, Koiwa H, Zhu J K, Hasegawa P M, Bressan R A. OSM1/SYP61: A syntaxin protein in Arabidopsis controls abscisic acid-mediated and non-abscisic acid-mediated responses to abiotic stress. The Plant Cell, 2002, 14: 3009-3028.

[18]Leshem Y, Melamed-Book N, Cagnac O, Ronen G, Nishri Y, Solomon M, Cohen G, Levine A. Suppression of Arabidopsis vesicle-SNARE expression inhibited fusion of H2O2 containing vesicles with tonoplast and increased salt tolerance. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(47): 18008-18013.

[19]Leshem Y, Golani Y, Kaye Y, Levine A. Reduced expression of the v-SNAREs AtVAMP71/AtVAMP7C gene family in Arabidopsis reduces drought tolerance by suppression of abscisic acid-dependent stomatal closure. Journal of Experimental Botany, 2010, 61(10): 2615-2622.

[20]Hamaji K, Nagira M, Yoshida K, Ohnishi M, Oda Y, Uemura T, Goh T, Sato M H, Morita M T, Tasaka M, Hasezawa S, Nakano A, Hara-Nishimura I, Maeshima M, Fukaki H, Mimura T. Dynamic aspects of ion accumulation by vesicle traffic under salt stress in Arabidopsis. Plant Cell and Physiology, 2009, 50(12): 2023-2033.

[21]范虎, 赵团结, 丁艳来, 邢光南, 盖钧镒. 中国野生大豆群体特征和地理分化的遗传分析. 中国农业科学, 2012, 45(3): 414-425.

Fan H, Zhao T J, Ding Y L, Xing G N, Gai J Y. Genetic analysis of the characteristics and geographic differentiation of Chinese wild soybean population. Scientia Agricultura Sinica, 2012, 45(3): 414-425. (in Chinese)

[22]王吴彬, 何庆元, 杨红燕, 向仕华, 赵团结, 邢光南, 盖钧镒. 大豆分枝数和叶柄夹角的相关野生片段分析. 中国农业科学, 2012, 45(23): 4749-4758. Wang W B, He Q Y, Yang H Y, Xiang S H, Zhao T J, Xing G N, Gai J Y. Detection of wild segments associated with number of branches on main stem and leafstalk angle in soybean. Scientia Agricultura Sinica, 2012, 45(23): 4749-4758.

[23]Zhang L, Zhang H, Liu P, Hao H, Jin J B, Lin J. Arabidopsis R-SNARE proteins VAMP721 and VAMP722 are required for cell plate formation. PloS ONE, 2011, 6(10): e26129.

[24]Yang L, Ji W, Zhu Y, Gao P, Li Y, Cai H, Bai X, Guo D. GsCBRLK, a calcium/calmodulin-binding receptor- like kinase, is a positive regulator of plant tolerance to salt and ABA stress. Journal of Experimental Botany, 2010, 61(9): 2519-2533.

[25]Mengiste T, Chen X, Salmeron J, Dietrich R. The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3 MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis. The Plant Cell, 2003, 11: 2551-2565.

[26]Jung C, Seo J S, Han S W, KooY J, Kim C H, Song S I, Nahm B H, Choi Y D, Cheong J. Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant Physiology, 2008, 146(2): 623-635.

[27]Chinnusamy V, Schumaker K, Zhu J. Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. Journal of Experimental Botany, 2004, 55(395): 225-236.

[28]Simpson S D, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence. The Plant Journal, 2003, 33: 259-270.

[29]Kaplan B, Davydov O, Knight H, Galon Y, Knight M R, Fluhr R, Fromm H. Rapid transcriptome changes induced by cytosolic Ca2+ transients reveal ABRE-related sequences as Ca2+-responsive cis elements in Arabidopsis. The Plant Cell, 2006, 18: 2733-2748.

[30]Dubouzet J G, Sakuma Y, Ito Y, Kasuga M, Dubouzet E G, Miura S, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. The Plant Journal, 2003, 33: 751-763.

[31]Busk P K, Jensen A B, Pages M. Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize. The Plant Journal, 1997, 11(6): 1285-1295.

[32]Park H C, Kim M L, Kang Y H, Jeon J M, Yoo J H, Kim M C, Park C Y, Jeong J C, Moon B C, Lee J H, Yoon H W, Lee S, Chung W S, Lim C O, Lee S Y, Hong J C, Cho M J. Pathogen- and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor. Plant Physiology, 2004, 135: 2150-2161.

[33]White A J, Dunn M A, Brown K, Hughes M A. Comparative analysis of genomic sequence and expression of a lipid transfer protein gene family in winter barley. Journal of Experimental Botany, 1994, 45: 1885-1892.

[34]Baker S S, Wilhelm K S, Thomashow M F. The 5′-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought and ABA-regulated gene expression. Plant Molecular Biology, 1994, 24: 701-713.

[35]Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. The Plant Cell, 2003, 15: 63-78.

[36]Luscher B, Eiseman R N. New light on Myc and Myb. Part II. Myb. Genes Development, 1992, 4: 2235-2241.

[37]王臻昱, 才华, 柏锡, 纪巍, 李勇, 魏正巍, 朱延明. 野生大豆GsGST19基因的克隆及其转基因苜蓿的耐盐碱性分析. 作物学报, 2012, 38(6): 971-979.

Wang Z Y, Cai H, Bai X, Ji W, Li Y, Wei Z W, Zhu Y M. Isolation of GsGST19 from Glycine soja and analysis of saline-alkaline tolerance for transgenic Medicago sativa. Acta Agronomica Sinica, 2012, 38(6): 971-979. (in Chinese)

[38]朱丹, 柏锡, 朱延明, 才华, 李勇, 纪巍, 陈超, 安琳, 朱毅. 野生大豆盐碱胁迫相关GsTIFY11b的克隆与功能分析. 遗传, 2012, 34(2): 230-239.

Zhu D, Bai X, Zhu Y M, Cai H, Li Y, Ji W, Chen C, An L, Zhu Y. Isolation and functional analysis of GsTIFY11b relevant to salt and alkaline stress from Glycine soja. Hereditas, 2012, 34(2): 230-239. (in Chinese)

[39]王果, 胡正, 张保缺, 王成社, 张辉. 山西省野生大豆资源遗传多样性分析. 中国农业科学, 2008, 41(7): 2182-2190.

Wang G, Hu Z, Zhang B Q, Wang C S, Zhang H. Genetic diversity analysis of Shanxi’s wild soybean (Glycine soja). Scientia Agricultura Sinica, 2008, 41(7): 2182-2190. (in Chinese)

[40]邱丽娟, 王昌陵, 周国安, 陈受宜, 常汝镇. 大豆分子育种研究进展. 中国农业科学, 2007, 40(11): 2418-2436.

Qiu L J, Wang C L, Zhou G A, Chen S Y, Chang R Z. Soybean molecular breeding. Scientia Agricultura Sinica, 2007, 40(11): 2418-2436. (in Chinese)

[41]Fu J, Naren A P, Gao X, Ahmmed G U, Malik A B. Protease- activated receptor-1 activation of endothelial cells induces protein kinase Ca-dependent phosphorylation of syntaxin 4 and Munc18c. The Journal of Biological Chemistry, 2005, 280(5): 3178-3184.

[42]Shuang R, Zhang L, Fletcher A, Groblewski G E, Pevsner J, Stuenkel E L. Regulation of Munc-18/Syntaxin 1A interaction by cyclin-dependent kinase 5 in nerve endings. The Journal of Biological Chemistry, 1998, 273(9): 4957-4966.

[43]Matveeva E A, Whiteheart S W, Vanaman T C, Slevin J T. Phosphorylation of the N-ethylmaleimide-sensitive factor is associated with depolarization-dependent neurotransmitter release from synaptosomes. The Journal of Biological Chemistry, 2001, 276(15): 12174-12181.

[44]谭勋, 刘艳娟, 潘家强, 李锦春, 孙卫东, 王小龙. 低温诱导的肺动脉高压肉鸡肺细小动脉PKCα 表达变化及其与肺血管重构的关系. 中国农业科学, 2005, 38(9): 1917-1922.

Tan X, Liu Y J, Pan J Q, Li J C, Sun W D, Wang X L. Expression of PKCα in pulmonary arterioles and its association with pulmonary vascular remodeling in broilers with pulmonary hypertension induced by cold temperature exposure. Scientia Agricultura Sinica, 2005, 38(9): 1917-1922. (in Chinese)

[45]Chheda M G, Ashery U, Thakur P, Rettig J, Sheng Z H. Phosphorylation of Snapin by PKA modulates its interaction with the SNARE complex. Nature Cell Biology, 2001, 3: 331-338.

[46]杨红, 朱利泉, 张贺翠, 杨永军, 薛丽琰, 杨昆, 余浩, 彭一波, 罗兵, 吴志刚, 黄丹, 高启国, 王小佳. 利用酵母双杂交系统鉴定甘蓝SCR与SRK胞外域片段间的相互作用. 中国农业科学, 2011, 44(9):1953-1962.

Yang H, Zhu L Q, Zhang H C, Yang Y J, Xue L Y, Yang K, Yu H, Peng Y B, Luo B, Wu Z G, Huang D, Gao Q G, Wang X J. Study on the interactions between the truncated fragments of SCR and eSRK from Brassica oleracea L. by a yeast two-hybrid system. Scientia Agricultura Sinica, 2011, 44(9): 1953-1962. (in Chinese)

[47]张春晓, 王文棋, 蒋湘宁, 陈雪梅. 植物基因启动子研究进展. 遗传学报, 2004, 31(12): 1455-1464.

Zhang C X, Wang W Q, Jiang X N, Chen X M. Review on plant gene promoters. Acta Genetica Sinica, 2004, 31(12): 1455-1464. (in Chinese)

[48]张利娟, 梁卫红, 刘悦霞, 刘肖飞, 侯成千, 毕佳佳. 两种水稻OsRhoGDIs基因启动子的克隆及分析. 中国农业科学. 2008, 41(10): 2916-2922.

Zhang L J, Liang W H, Liu Y X, Liu X F, Hou C Q, Bi J J. Cloning and analysis of the promoters of two OsRhoGDIs genes in rice. Scientia Agricultura Sinica, 2008, 41(10): 2916-2922. (in Chinese)

[49]Hamaji K, Nagira M, Yoshida K, Ohnishi M, Oda Y, Uemura T, Goh T, Sato M H, Morita M T, Tasaka M, Hasezawa S, Nakano A, Hara-Nishimura I, Maeshima M, Fukaki H, Mimura T. Dynamic aspects of ion accumulation by vesicle traffic under salt stress in Arabidopsis. Plant Cell and Physiology, 2009, 50(12): 2023-2033.

[50]Lawrence R J, Pikaard C S. Transgene-induced RNA interference: A strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations. The Plant Journal, 2003, 36(1): 114-121.

[51]王昌陵, 赵军, 李英慧, 范云六, 张丽娟, 刘章雄, 关荣霞, 吕淑霞, 常汝镇, 邱丽娟. 转录因子ABP9转化大豆 (Glycine max L.)及遗传转化条件优化. 中国农业科学, 2008, 41(7): 1908-1916.

Wang C L, Zhao J, Li Y H, Fan Y L, Zhang L J, Liu Z X, Guan R X, Lü S X, Chang R Z, Qiu L J. Transforming transcription factor ABP9 into soybean and optimization of the transformation system. Scientia Agricultura Sinica, 2008, 41(7): 1908-1916. (in Chinese)