棉花CML基因家族成员鉴定与功能分析
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摘要
【目的】类钙调素(Calmodulin-like,CML)蛋白是1种重要的Ca2+传感器,在调节植物应激反应机制中起重要作用。对CML基因家族进行全基因组学分析,以便深入研究CML基因在棉花逆境胁迫下的作用。【方法】利用生物信息学的方法进行了棉花CML家族成员的鉴定。利用转录组数据和反转录聚合酶链式反应分析陆地棉(Gossypium hirsutum L.)CML基因在盐胁迫下的表达模式。利用病毒诱导基因沉默技术对Gh CML44-2基因进行沉默并进行功能验证。【结果】在陆地棉(Gossypium hirsutum L.)、雷蒙德氏棉(G. raimondii Ulbrich)、亚洲棉(G. arboreum L.)中分别获得了154个、74个、78个CML蛋白。进化树和保守基序分析表明,GhCML蛋白分为8个亚类,都含有保守的EF-hand结构域;在盐胁迫下,107个Gh CML基因在叶和根中有显著的差异表达,且在根和叶中的差异表达模式不同。启动子分析表明,这些基因启动子中存在多种不同的胁迫响应元件;利用病毒诱导基因沉默技术成功沉默Gh CML44-2的棉株相较于对照更加不耐盐。【结论】该研究结果有助于了解棉花CML基因家族的进化与功能,为后续研究其功能提供了一定的理论依据。
[Objective] Calmodulin-like protein(CML) is an important Ca2+sensor and plays an important role in regulating plant stress response mechanism. Genome-wide analysis of CML gene family provides a basis for further study of the role of CML genes in cotton under stress. [Method] The members of CML family in cotton were identified by bioinformatics. The expression patterns of CML genes in upland cotton under salt stress were analyzed by the transcriptome data and real-time reverse transcription-polymerase chain reaction. Virus-induced gene silencing(VIGS) was used to silence Gh CML44-2 gene and to verify its function. [Result] 154, 74 and 78 CML proteins were obtained from Gossypium hirsutum L., G. raimondii Ulbrich and G. arboreum L., respectively. Evolutionary tree and conservative motif analysis showed that GhCML proteins were divided into eight subgroups, all containing conserved EF-hand domain. Under salt stress, 107 GhCML genes were differentially expressed with different expression patterns in leaves and roots. Promoter analysis showed that there were many different stress response elements in these gene promoters, and cotton plants were more salt-tolerant than control plants after silencing the expression of Gh CML44-2 successfully by using VIGS. [Conclusion] The results of this study are helpful to understand the evolution and function of CML gene family in cotton, and provide a theoretical basis and reference for the follow-up study of their specific functions in cotton.
[Objective] Calmodulin-like protein(CML) is an important Ca2+sensor and plays an important role in regulating plant stress response mechanism. Genome-wide analysis of CML gene family provides a basis for further study of the role of CML genes in cotton under stress. [Method] The members of CML family in cotton were identified by bioinformatics. The expression patterns of CML genes in upland cotton under salt stress were analyzed by the transcriptome data and real-time reverse transcription-polymerase chain reaction. Virus-induced gene silencing(VIGS) was used to silence Gh CML44-2 gene and to verify its function. [Result] 154, 74 and 78 CML proteins were obtained from Gossypium hirsutum L., G. raimondii Ulbrich and G. arboreum L., respectively. Evolutionary tree and conservative motif analysis showed that GhCML proteins were divided into eight subgroups, all containing conserved EF-hand domain. Under salt stress, 107 GhCML genes were differentially expressed with different expression patterns in leaves and roots. Promoter analysis showed that there were many different stress response elements in these gene promoters, and cotton plants were more salt-tolerant than control plants after silencing the expression of Gh CML44-2 successfully by using VIGS. [Conclusion] The results of this study are helpful to understand the evolution and function of CML gene family in cotton, and provide a theoretical basis and reference for the follow-up study of their specific functions in cotton.
引文
[1] Huang C, Nie X, Shen C, et al. Population structure and genetic basis of the agronomic traits of upland cotton in China revealed by a genome‐wide association study using high‐density SNPs[J]. Plant Biotechnology Journal, 2017, 15(11):1374-1386.
[2] Liu J, Guo W Q, Shi D C. Seed germination, seedling survival,and physiological response of sunflowers under saline and alkaline conditions[J]. Photosynthetica, 2010, 48(2):278-286.
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[5] Deng F, Zhang X, Wang W, et al. Identification of Gossypium hirsutum long non-coding RNAs(lncRNAs)under salt stress[J].BMC Plant Biology, 2018, 18(1):23. https://doi.org/10.1186/s12870-018-1238-0
[6] Sanchez-Barrena M J, Martinez-Ripoll M, Zhu J K, et al. SOS3(salt overly sensitive 3)from Arabidopsis thaliana:expression,purification, crystallization and preliminary X-ray analysis[J].Acta Crystallographica Section D:Biological Crystallography,2004, 60(7):1272-1274.
[7] Zhu J K. Abiotic stress signaling and responses in plants[J]. Cell,2016, 167(2):313-324.
[8] Midhat U, Ting M K Y, Teresinski H J, et al. The calmodulin-like protein, CML39, is involved in regulating seed development, germination, and fruit development in Arabidopsis[J].Plant Molecular Biology, 2018, 96(4/5):1-18.
[9] Bender K W, Snedden W A. Calmodulin-related proteins step out from the shadow of their namesake[J]. Plant Physiology,2013, 163(2):486-495.
[10] Edel K H, Kudla J. Increasing complexity and versatility:How the calcium signaling toolkit was shaped during plant land colonization[J]. Cell Calcium, 2015, 57(3):231-246.
[11] Zhu X, Dunand C, Snedden W, et al. Ca M and CML emergence in the green lineage[J]. Trends in Plant Science, 2015, 20(8):483-489.
[12] Defalco T A, Chiasson D, Munro K, et al. Characterization of GmCaMK1, a member of a soybean calmodulin-binding receptor-like kinase family[J]. FEBS Letters, 2010, 584(23):4717-4724.
[13] Kretsinger R H, Nockolds C E. Carp muscle calcium-binding protein. II. Structure determination and general description[J].Journal of Biological Chemistry, 1973, 248(9):3313-3326.
[14] Poovaiah B W, Du L, Wang H, et al. Recent advances in calcium/calmodulin-mediated signaling with an emphasis on plant-microbe interactions[J]. Plant Physiology, 2013, 163(2):531-542.
[15] Delk N A, Johnson K A, Chowdhury N I, et al. CML24, regulated in expression by diverse stimuli, encodes a potential Ca2+sensor that functions in responses to abscisic acid, daylength,and ion stress[J]. Plant Physiology, 2005, 139(1):240-253.
[16] Boonburapong B, Buaboocha T. Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins[J]. BMC Plant Biology, 2007, 7(1):4. https://doi.org/10.1186/1471-2229-7-4
[17] Munir S, Khan M R G, Song J, et al. Genome-wide identification, characterization and expression analysis of calmodulin-like(CML)proteins in tomato(Solanum lycopersicum)[J].Plant Physiology and Biochemistry, 2016, 102:167-179.
[18]陈超,端木慧子,朱丹,等.大豆CML家族基因的生物信息学分析[J].大豆科学, 2015, 34(6):957-963.Chao Chao, Duammu Huizi, Zhu Dan, et al. Bioinformatics analysis of GmCML genes in soybean genome[J]. Soybean Science, 2015, 34(6):957-963.
[19] Vanderbeld B, Snedden W A. Developmental and stimulus-induced expression patterns of Arabidopsis calmodulin-like genes CML37, CML38 and CML39[J]. Plant Molecular Biology,2007, 64(6):683-697.
[20] Scholz S S, Reichelt M, Vadassery J, et al. Calmodulin-like protein CML37 is a positive regulator of ABA during drought stress in Arabidopsis[J]. Plant Signaling&Behavior, 2015, 10(6):e1011951. https://doi.org/10.1080/15592324.2015.1011951
[21] Hirai M Y, Sawada Y, Kanaya S, et al. Toward genome-wide metabolotyping and elucidation of metabolic system:metabolic profiling of large-scale bioresources[J]. Journal of Plant Research, 2010, 123(3):291-298.
[22] Magnan F, Ranty B, Charpenteau M, et al. Mutations in AtCML9, a calmodulin‐like protein from Arabidopsis thaliana,alter plant responses to abiotic stress and abscisic acid[J]. The Plant Journal, 2008, 56(4):575-589.
[23] Xu G Y, Rocha P S C F, Wang M L, et al. A novel rice calmodulin-like gene, Os MSR2, enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis[J]. Planta, 2011,234(1):47-59.
[24] Voorrips R E. Map Chart:Software for the graphical presentation of linkage maps and QTLs[J]. Journal of Heredity, 2002,93(1):77-78.
[25] Perochon A, Aldon D, Galaud J P, et al. Calmodulin and calmodulin-like proteins in plant calcium signaling[J].Biochimie, 2011, 93(12):2048-2053. https://doi.org/10.1016/j.biochi.2011.07.012
[26] Day I S, Reddy V S, Ali G S, et al. Analysis of EF-hand-containing proteins in Arabidopsis[J]. Genome Biology, 2002, 3(10):research0056.1. https://doi.org/10.1186/gb-2002-3-10-research0056
[27] Mccormack E, Braam J. Calmodulins and related potential calcium sensors of Arabidopsis[J]. New Phytologist, 2010, 159(3):585-598.
[28] Ali G S, Reddy V S, Lindgren P B, et al. Differential expression of genes encoding calmodulin-binding proteins in response to bacterial pathogens and inducers of defense responses[J]. Plant Molecular Biology, 2003, 51(6):803-815.
[29] Liu H T, Li B, Shang Z L, et al. Calmodulin is involved in heat shock signal transduction in wheat[J]. Plant Physiology, 2003,132(3):1186-1195.
[30] Reddy A S N. Calcium:Silver bullet in signaling[J]. Plant Science, 2001, 160(3):381-404.
[31] Snedden W A, Fromm H. Calmodulin as a versatile calcium signal transducer in plants[J]. New Phytologist, 2001, 151(1):35-66.
[32]Cheng H Q, Han L B, Yang C L, et al. The cotton MYB108forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection[J]. Journal of Experimental Botany, 2016, 67(6):1935-1950.
[2] Liu J, Guo W Q, Shi D C. Seed germination, seedling survival,and physiological response of sunflowers under saline and alkaline conditions[J]. Photosynthetica, 2010, 48(2):278-286.
[3] Munns R, Tester M. Mechanisms of salinity tolerance[J]. Annual Review of Plant Biology, 2008, 59(1):651-681.
[4] Shavrukov Y, Langridge P, Tester M. Salinity tolerance and sodium exclusion in genus Triticum[J]. Breeding Science, 2009, 59(5):671-678.
[5] Deng F, Zhang X, Wang W, et al. Identification of Gossypium hirsutum long non-coding RNAs(lncRNAs)under salt stress[J].BMC Plant Biology, 2018, 18(1):23. https://doi.org/10.1186/s12870-018-1238-0
[6] Sanchez-Barrena M J, Martinez-Ripoll M, Zhu J K, et al. SOS3(salt overly sensitive 3)from Arabidopsis thaliana:expression,purification, crystallization and preliminary X-ray analysis[J].Acta Crystallographica Section D:Biological Crystallography,2004, 60(7):1272-1274.
[7] Zhu J K. Abiotic stress signaling and responses in plants[J]. Cell,2016, 167(2):313-324.
[8] Midhat U, Ting M K Y, Teresinski H J, et al. The calmodulin-like protein, CML39, is involved in regulating seed development, germination, and fruit development in Arabidopsis[J].Plant Molecular Biology, 2018, 96(4/5):1-18.
[9] Bender K W, Snedden W A. Calmodulin-related proteins step out from the shadow of their namesake[J]. Plant Physiology,2013, 163(2):486-495.
[10] Edel K H, Kudla J. Increasing complexity and versatility:How the calcium signaling toolkit was shaped during plant land colonization[J]. Cell Calcium, 2015, 57(3):231-246.
[11] Zhu X, Dunand C, Snedden W, et al. Ca M and CML emergence in the green lineage[J]. Trends in Plant Science, 2015, 20(8):483-489.
[12] Defalco T A, Chiasson D, Munro K, et al. Characterization of GmCaMK1, a member of a soybean calmodulin-binding receptor-like kinase family[J]. FEBS Letters, 2010, 584(23):4717-4724.
[13] Kretsinger R H, Nockolds C E. Carp muscle calcium-binding protein. II. Structure determination and general description[J].Journal of Biological Chemistry, 1973, 248(9):3313-3326.
[14] Poovaiah B W, Du L, Wang H, et al. Recent advances in calcium/calmodulin-mediated signaling with an emphasis on plant-microbe interactions[J]. Plant Physiology, 2013, 163(2):531-542.
[15] Delk N A, Johnson K A, Chowdhury N I, et al. CML24, regulated in expression by diverse stimuli, encodes a potential Ca2+sensor that functions in responses to abscisic acid, daylength,and ion stress[J]. Plant Physiology, 2005, 139(1):240-253.
[16] Boonburapong B, Buaboocha T. Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins[J]. BMC Plant Biology, 2007, 7(1):4. https://doi.org/10.1186/1471-2229-7-4
[17] Munir S, Khan M R G, Song J, et al. Genome-wide identification, characterization and expression analysis of calmodulin-like(CML)proteins in tomato(Solanum lycopersicum)[J].Plant Physiology and Biochemistry, 2016, 102:167-179.
[18]陈超,端木慧子,朱丹,等.大豆CML家族基因的生物信息学分析[J].大豆科学, 2015, 34(6):957-963.Chao Chao, Duammu Huizi, Zhu Dan, et al. Bioinformatics analysis of GmCML genes in soybean genome[J]. Soybean Science, 2015, 34(6):957-963.
[19] Vanderbeld B, Snedden W A. Developmental and stimulus-induced expression patterns of Arabidopsis calmodulin-like genes CML37, CML38 and CML39[J]. Plant Molecular Biology,2007, 64(6):683-697.
[20] Scholz S S, Reichelt M, Vadassery J, et al. Calmodulin-like protein CML37 is a positive regulator of ABA during drought stress in Arabidopsis[J]. Plant Signaling&Behavior, 2015, 10(6):e1011951. https://doi.org/10.1080/15592324.2015.1011951
[21] Hirai M Y, Sawada Y, Kanaya S, et al. Toward genome-wide metabolotyping and elucidation of metabolic system:metabolic profiling of large-scale bioresources[J]. Journal of Plant Research, 2010, 123(3):291-298.
[22] Magnan F, Ranty B, Charpenteau M, et al. Mutations in AtCML9, a calmodulin‐like protein from Arabidopsis thaliana,alter plant responses to abiotic stress and abscisic acid[J]. The Plant Journal, 2008, 56(4):575-589.
[23] Xu G Y, Rocha P S C F, Wang M L, et al. A novel rice calmodulin-like gene, Os MSR2, enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis[J]. Planta, 2011,234(1):47-59.
[24] Voorrips R E. Map Chart:Software for the graphical presentation of linkage maps and QTLs[J]. Journal of Heredity, 2002,93(1):77-78.
[25] Perochon A, Aldon D, Galaud J P, et al. Calmodulin and calmodulin-like proteins in plant calcium signaling[J].Biochimie, 2011, 93(12):2048-2053. https://doi.org/10.1016/j.biochi.2011.07.012
[26] Day I S, Reddy V S, Ali G S, et al. Analysis of EF-hand-containing proteins in Arabidopsis[J]. Genome Biology, 2002, 3(10):research0056.1. https://doi.org/10.1186/gb-2002-3-10-research0056
[27] Mccormack E, Braam J. Calmodulins and related potential calcium sensors of Arabidopsis[J]. New Phytologist, 2010, 159(3):585-598.
[28] Ali G S, Reddy V S, Lindgren P B, et al. Differential expression of genes encoding calmodulin-binding proteins in response to bacterial pathogens and inducers of defense responses[J]. Plant Molecular Biology, 2003, 51(6):803-815.
[29] Liu H T, Li B, Shang Z L, et al. Calmodulin is involved in heat shock signal transduction in wheat[J]. Plant Physiology, 2003,132(3):1186-1195.
[30] Reddy A S N. Calcium:Silver bullet in signaling[J]. Plant Science, 2001, 160(3):381-404.
[31] Snedden W A, Fromm H. Calmodulin as a versatile calcium signal transducer in plants[J]. New Phytologist, 2001, 151(1):35-66.
[32]Cheng H Q, Han L B, Yang C L, et al. The cotton MYB108forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection[J]. Journal of Experimental Botany, 2016, 67(6):1935-1950.