盐地碱蓬AGO蛋白基因家族的分离及表达分析
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摘要
AGO蛋白(Argonature protein)作为RNA诱导沉默复合物的核心元件,在植物的非生物胁迫过程中发挥着重要作用。以前期获得的盐地碱蓬转录组数据为基础,开展了针对盐地碱蓬(Suaeda salsa)AGO蛋白的生物信息学及盐胁迫下的表达规律研究。采用RACE技术克隆2个AGO基因家族成员(SsAGO1、SsAGO16)的cDNA全长序列,并对其蛋白结构、进化关系、表达模式等进行分析。结果表明:盐地碱蓬2类AGO蛋白家族成员基因长度分别为3 212 bp和3 766 bp,编码的蛋白均是一个无跨膜结构域、无信号肽的亲水性蛋白,α-螺旋散布于每个蛋白质的整体结构中;SsAGO1和SsAGO16都含有一个未知的结构域DUF1785,除此之外,SsAGO1还含有PAZ和PIWI两个特征结构域,而SsAGO16仅含有PIWI结构域,SsAGO1和SsAGO16亲缘关系较远,且具有不同的时空表达模式。首次从盐地碱蓬鉴定了AGO蛋白基因并进行了盐胁迫下的表达规律分析,为研究AGO蛋白家族在盐生植物耐盐生理过程中的作用奠定了基础。
Argonaute protein(AGO) is the core effector in RNA-induced silencing complexes(RISCs), playing an important role in abiotic stresses in plants. Based on the transcriptome data of Suaeda salsa, we studied the bioinformatics and expression pattern of AGO protein family under the salt stress. Full-length cDNA sequences of two AGO genes(SsAGO1 and SsAGO16) were cloned by RACE technique, and their genetic structure, evolutionary relationship and expression pattern were analyzed. The results show that two AGO genes contain 3 212 bp and 3 766 bp respectively, encoding a hydrophilic protein without transmembrane domain and signal peptide, and it possesses lots of alphahelix in each protein. Both SsAGO1 and SsAGO16 contain an unknown domain DUF1785. In addition, SsAGO1 contains PAZ and PIWI domains, and SsAGO16 contains PIWI domain. SsAGO1 and SsAGO16 have a far distant relationship and have different temporal and spatial expression patterns. In this paper, these two AGO genes were identified for the first time from Suaeda salsa and the expression was analyzed under salt stress, which laid a foundation for further research on the role of AGO gene family in salt tolerance physiology of halophytes.
Argonaute protein(AGO) is the core effector in RNA-induced silencing complexes(RISCs), playing an important role in abiotic stresses in plants. Based on the transcriptome data of Suaeda salsa, we studied the bioinformatics and expression pattern of AGO protein family under the salt stress. Full-length cDNA sequences of two AGO genes(SsAGO1 and SsAGO16) were cloned by RACE technique, and their genetic structure, evolutionary relationship and expression pattern were analyzed. The results show that two AGO genes contain 3 212 bp and 3 766 bp respectively, encoding a hydrophilic protein without transmembrane domain and signal peptide, and it possesses lots of alphahelix in each protein. Both SsAGO1 and SsAGO16 contain an unknown domain DUF1785. In addition, SsAGO1 contains PAZ and PIWI domains, and SsAGO16 contains PIWI domain. SsAGO1 and SsAGO16 have a far distant relationship and have different temporal and spatial expression patterns. In this paper, these two AGO genes were identified for the first time from Suaeda salsa and the expression was analyzed under salt stress, which laid a foundation for further research on the role of AGO gene family in salt tolerance physiology of halophytes.
引文
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[12] ZHAO L,XIA W Q,CAI X Z.Identification of subcellular localization of Arabidopsis AGO2[J].Journal of Zhejiang University (Agriculture and Life Science),2013,39(1):1-10.赵丽,夏文强,蔡新忠.拟南芥AGO2的亚细胞定位分析[J].浙江大学学报(农业与生命科学),2013,39(1):1-10.
[13] LI C,DU Z Y,CHEN J S.Structural and functional elucidation of AGO proteins[J].Chinese Journal of Biochemistry and Molecular Biology,2009,25(11):969-976.李超,杜志游,陈集双.解读AGO蛋白结构及其功能[J].中国生物化学与分子生物学报,2009,25 (11) :969-976.
[14] MOREL J B,GODON C,MOURRAIN P,et al.Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance[J].Plant Cell,2002,14(3):629-639.
[15] KAPOOR M,ARORA R,LAMA T,et al.Genome-wide identification,organization and phylogenetic analysis of Dicer-like,Argonaute and RNA-dependent RNA polymerase gene families and their expression analysis during reproductive development and stress in rice[J].BMC Genomics,2008,9(1):451.
[16] MST B,KAZI H,RANJAN S,et al.Pea p68 imparts salinity stress tolerance in rice by scavenging of ROS-mediated H2O2,and interacts with Argonaute[J].Plant Molecular Biology Reporter,2014,33(2):221-238.
[17] ZHANG A Q,PANG Q Y,YAN X F.Advances in salt-tolerance mechanisms of Suaeda plant[J].Acta Ecologica Sinica,2013,33(12):3575-3583.张爱琴,庞秋颖,阎秀峰.碱蓬属植物耐盐机理研究进展[J].生态学报,2013,33(12):3575-3583.
[18] PENG B,XU W,SHAO R,et al.Growth of Suaeda salsa tin response to salt stress in different habitats[J].Acta Prataculturae Sinica,2016,25(4):81-90.彭斌,许伟,邵荣,等.不同生境种源盐地碱蓬幼苗生长发育对盐分胁迫的响应和适应[J].草业学报,2016,25(4):81-90.
[19] ZHANG X,FAN S,LI F.Development and utilization of Suaede salsa in China[J].Chinese Wild Plant Resources,2003,22(2):1-3.
[20] BORGES F,PEREIRA P A,Slotkin R K,et al.MicroRNA activity in the Arabidopsis male germline[J].Journal of Experimental Botany,2011,62(5):1611-1620.
[21] ZOU Y,TANG H.Roles of Argonaute proteins in RNA interference[J].Journal of Molecular Biology,2006,3(1):55-57.邹扬,汤华.Argonaute 蛋白家族在RNAi中的作用[J].医学分子生物学杂志,2006,3(1):55-57.
[22] LAW J A,JACOBSEN S E.Establishing,maintaining and modifying DNA methylation patterns in plant and animals[J].Nature Reviews Genetics,2010,11(3):204-220.
[23] BAI M,YANG G S,CHEN W T,et al.Genome-wide identification of Dicer-like,Argonaute and RNA-dependent RNA polymerase gene families and their expression analyses in response to viral infection and abiotic stresses in Solanum lycopersicum[J].Gene,2012,501 (1) :52-62.
[24] YUE L M,SONG J B,XU X F,et al.Bioinformatical and experimental analysis of AGO genes in response to salt stress[J].Journal of Shenzhen University (Science & Engineering),2017,34(4):352-357.岳路明,宋剑波,徐晓峰,等.拟南芥AGO基因家族分析及盐胁迫下的表达验证[J].深圳大学学报 (理工版),2017,34(4):352-357.
[25] CHERIAN S,REDDY M P.Evaluation of NaCl tolerance in the callus cultures of Suaeda nudiflora Moq.[J].Biologia plantarum,2003,46(2):193-198.
[26] BALNOKIN Y,KURKOVA E,Myasoedov N,et al.Structural and functional state of thylakoids in a halophyte Suaeda altissima before and after disturbance of salt-water balance by extremely high concentrations of NaCl[J].Russian Journal of Plant Physiology,2004,51(6):815-821.
[2] CAI J,SHANG H H,LI W J,et al.Bioinformatics analysis of argonaute protein family in gossypium arboretum[J].Journal of Northwest A & F University (Natural Science Edition),2017,45(1):61-69.蔡娟,商海红,李伟杰,等.亚洲棉Argonaute蛋白家族的生物信息学预测与分析[J].西北农林科技大学学报 (自然科学版),2017,45(1):61-69.
[3] LI L,XUE X,ZUO S M,et al.Suppressed expression of OsAGO1a leads to adaxial leaf rolling in rice[J].Chinese Journal of Rice Science,2013,27(3):223-230.李磊,薛芗,左示敏,等.抑制OsAGO1a基因的表达导致水稻叶片近轴面卷曲[J].中国水稻科学,2013,27(3):223-230.
[4] BAUMBERGER N,BAULCOMBE D C.Arabidopsis ARGONAUTE1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs[J].Proceedings of the National Academy of Sciences,2005,102(33):11928-11933.
[5] HUTVAGNER G,SIMARD M J.Argonaute proteins:key players in RNA silencing[J].Nature Reviews Molecular Cell Biology,2008,9(9):22-32.
[6] DARRICARRERE N,LIU N,WATANABE T,et al.Function of Piwi,a nuclear Piwi/Argonaute protein,is independent of its slicer activity[J].Proceedings of the national academy of sciences of the united states of america,2013,110(4):1279-1302.
[7] YIGIT E,BATISTA P J,BEI Y,et al.Analysis of the C.elegans Argonaute family reveals that distinct argonautes act sequentially during RNAi[J].Cell,2006,127(4):747-757.
[8] KWAK P B,TOMARI Y.The N domain of Argonaute drives duplex unwinding during RISC assembly[J].Nature Structural and Molecular Biology,2012,19:145-151.
[9] FRANK F,SONENBERG N,NAGAR B.Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2[J].Nature,2010,465(7299):818-822.
[10] WANG W J,LIU T,GUO L,et al.SLC/AGO1 coordinates cell division and expasnsionin Arabidopsis[J].Journal of Intergrative Plant Biology,2011,46(4):370-378.王文婧,刘婷,郭磊,等.SLC/AGO1基因控制拟南芥细胞分裂与定向伸长[J].植物学报,2011,46(4):370-378.
[11] TANG Z H,LIU Y B,QUAN Z W.Expression and function of Argonaute Critical Proteins in human tumor[J].Chinese Journal of Exoerimental Surgery,2010,27(12):1967-1968.汤朝晖,刘颖斌,全志伟.RNA基因沉默关键蛋白Argonaute在人类肿瘤中的表达及其作用[J].中华实验外科杂志,2010,27(12):1967-1968.
[12] ZHAO L,XIA W Q,CAI X Z.Identification of subcellular localization of Arabidopsis AGO2[J].Journal of Zhejiang University (Agriculture and Life Science),2013,39(1):1-10.赵丽,夏文强,蔡新忠.拟南芥AGO2的亚细胞定位分析[J].浙江大学学报(农业与生命科学),2013,39(1):1-10.
[13] LI C,DU Z Y,CHEN J S.Structural and functional elucidation of AGO proteins[J].Chinese Journal of Biochemistry and Molecular Biology,2009,25(11):969-976.李超,杜志游,陈集双.解读AGO蛋白结构及其功能[J].中国生物化学与分子生物学报,2009,25 (11) :969-976.
[14] MOREL J B,GODON C,MOURRAIN P,et al.Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance[J].Plant Cell,2002,14(3):629-639.
[15] KAPOOR M,ARORA R,LAMA T,et al.Genome-wide identification,organization and phylogenetic analysis of Dicer-like,Argonaute and RNA-dependent RNA polymerase gene families and their expression analysis during reproductive development and stress in rice[J].BMC Genomics,2008,9(1):451.
[16] MST B,KAZI H,RANJAN S,et al.Pea p68 imparts salinity stress tolerance in rice by scavenging of ROS-mediated H2O2,and interacts with Argonaute[J].Plant Molecular Biology Reporter,2014,33(2):221-238.
[17] ZHANG A Q,PANG Q Y,YAN X F.Advances in salt-tolerance mechanisms of Suaeda plant[J].Acta Ecologica Sinica,2013,33(12):3575-3583.张爱琴,庞秋颖,阎秀峰.碱蓬属植物耐盐机理研究进展[J].生态学报,2013,33(12):3575-3583.
[18] PENG B,XU W,SHAO R,et al.Growth of Suaeda salsa tin response to salt stress in different habitats[J].Acta Prataculturae Sinica,2016,25(4):81-90.彭斌,许伟,邵荣,等.不同生境种源盐地碱蓬幼苗生长发育对盐分胁迫的响应和适应[J].草业学报,2016,25(4):81-90.
[19] ZHANG X,FAN S,LI F.Development and utilization of Suaede salsa in China[J].Chinese Wild Plant Resources,2003,22(2):1-3.
[20] BORGES F,PEREIRA P A,Slotkin R K,et al.MicroRNA activity in the Arabidopsis male germline[J].Journal of Experimental Botany,2011,62(5):1611-1620.
[21] ZOU Y,TANG H.Roles of Argonaute proteins in RNA interference[J].Journal of Molecular Biology,2006,3(1):55-57.邹扬,汤华.Argonaute 蛋白家族在RNAi中的作用[J].医学分子生物学杂志,2006,3(1):55-57.
[22] LAW J A,JACOBSEN S E.Establishing,maintaining and modifying DNA methylation patterns in plant and animals[J].Nature Reviews Genetics,2010,11(3):204-220.
[23] BAI M,YANG G S,CHEN W T,et al.Genome-wide identification of Dicer-like,Argonaute and RNA-dependent RNA polymerase gene families and their expression analyses in response to viral infection and abiotic stresses in Solanum lycopersicum[J].Gene,2012,501 (1) :52-62.
[24] YUE L M,SONG J B,XU X F,et al.Bioinformatical and experimental analysis of AGO genes in response to salt stress[J].Journal of Shenzhen University (Science & Engineering),2017,34(4):352-357.岳路明,宋剑波,徐晓峰,等.拟南芥AGO基因家族分析及盐胁迫下的表达验证[J].深圳大学学报 (理工版),2017,34(4):352-357.
[25] CHERIAN S,REDDY M P.Evaluation of NaCl tolerance in the callus cultures of Suaeda nudiflora Moq.[J].Biologia plantarum,2003,46(2):193-198.
[26] BALNOKIN Y,KURKOVA E,Myasoedov N,et al.Structural and functional state of thylakoids in a halophyte Suaeda altissima before and after disturbance of salt-water balance by extremely high concentrations of NaCl[J].Russian Journal of Plant Physiology,2004,51(6):815-821.
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