盐胁迫下旱地棉叶片数字化基因表达谱分析
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摘要
为了深入研究棉花耐盐的分子机理,利用数字化基因表达谱技术,分析了盐胁迫处理3 h和72 h时旱地棉叶片中基因表达差异,分别获得1 778(1 046个上调、732个下调)和2 873(2 065个上调、808个下调)个差异表达基因。功能注释分析结果表明,这些差异表达基因主要参与了信号转导、逆境应答、能量代谢和转录调节等方面。随机挑选10个差异表达基因进行qRT-PCR验证,结果表明,qRT-PCR结果与数字化基因表达谱分析结果基本一致。
To uncover the salt tolerance mechanisms of Gossypium aridum and improve salinity resistance,the technique of digital gene expression profile was involved to analyze the variations of gene expression in the leaves of upland cotton exposed to 200 mmol/L Na Cl for 3 h and 72 h,respectively. There were 1 778( 1 046 up-regulated/732 down-regulated) and 2 873( 2 065 up-regulated/808down-regulated) differentially expressed genes,respectively. Functional annotation analysis revealed that the genes were involved in various metabolic pathways including signal transduction,stress response,energy metabolism,and transcriptional regulation. A total of 10 differentially expressed genes were randomly selected for qRT-PCR validation,and the results were basically consistent with those by the digital gene expression profile.
To uncover the salt tolerance mechanisms of Gossypium aridum and improve salinity resistance,the technique of digital gene expression profile was involved to analyze the variations of gene expression in the leaves of upland cotton exposed to 200 mmol/L Na Cl for 3 h and 72 h,respectively. There were 1 778( 1 046 up-regulated/732 down-regulated) and 2 873( 2 065 up-regulated/808down-regulated) differentially expressed genes,respectively. Functional annotation analysis revealed that the genes were involved in various metabolic pathways including signal transduction,stress response,energy metabolism,and transcriptional regulation. A total of 10 differentially expressed genes were randomly selected for qRT-PCR validation,and the results were basically consistent with those by the digital gene expression profile.
引文
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[23]LI W,GODZIK A.cd-hit:a fast program for clustering and comparing large sets of protein or nucleotide sequences[J].Bioinformatics,2006,22(13):1658-1659.
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[29]RODRIGUEZ-URIBE L,HIGBIE S M,STEWART J M,et al.Identification of salt responsive genes using comparative microarray analysis inupland cotton(Gossypium hirsutum L.)[J].Plant Science,2011,180:461-469.
[30]WANG G,ZHU Q G,MENG Q W,et al.Transcript profiling during salt stress of young cotton(Gossypium hirsutum)seedlings via Solexa sequencing[J].Acta Physiologiae Plantarum,2012,34(1):107.
[31]CHAUMONT F,BARRIEU F,WOJCIK E,et al.Aquaporins constitute a large and highly divergent protein family in maize[J].Plant Physiology,2001,125:1206-1215.
[32]KATSUHARA M,KOSHIO K,SHIBASAKA M,et al.Overexpression of a barley aquaporin incased the shoot/root ratio and raised salt sensitivity in transgenic rice plants[J].Plant and Cell Physiology,2003,44:1378-1383.
[33]XIANG Y,HUANG Y,XIONG L.Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement[J].Plant Physiology,2007,144:1416-1428.
[34]何亮,李富华,沙莉娜,等.玉米2C型丝氨酸/苏氨酸蛋白磷酸酶(PP2C)活性与耐旱性关系[J].作物学报,2008,34(5):899-903.
[35]MA H L,ZHOU H L,ZHANG H Y,et al.Cloning and expression analysis of an AP2/ERF gene and its responses to phytohormones and abiotic stresses in rice[J].Rice Science,2010,17:1-9.
[36]ZHU Q,ZHANG J,GAO X,et al.The Arabidopsis AP2/ERF transcription factor RAP2.6 participates in ABA,salt and osmotic stress responses[J].Gene,2010,457:1-12.
[2]WANG W,VINOCUR B,ALTMAN A.Plant responses to drought,salinity and extreme temperatures:towards genetic engineering for stress tolerance[J].Planta,2003,218:1-14.
[3]李刚波,李慧,丛郁,等.非生物胁迫下杜梨Pb CBL4基因的表达[J].江苏农业学报,2014,30(5):1132-1138.
[4]许奕,徐碧玉,胡伟,等.盐胁迫下Ma AQP1转基因拟南芥幼苗的生长和生理响应[J].江苏农业学报,2014,30(6):1279-1285.
[5]余璐璐,曹中权,刘龙山,等.盐芥CAS基因的生物信息学分析及在盐胁迫下的表达[J].江苏农业科学,2015,43(7):25-29.
[6]马沛勤.硫酸镁、复合盐胁迫下抗虫棉幼苗抗氧化生理反应[J].江苏农业科学,2014,42(2):59-61.
[7]卢军,娄伟,罗琳,等.低温和长时间贮藏对烟草种子萌发和幼苗渗透调节物质的影响[J].南方农业学报,2014,45(11):1943-1946.
[8]RAMACHANDRA-REDDY A,CHAITANYA K V,VIVEKANANDAN M.Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants[J].Journal of Plant Physiology,2004,161:1189-1202.
[9]ZHANG L,XI D M,LI S W,et al.A cotton group C MAP kinase gene,Gh MPK2 positively regulates salt and drought tolerance in tobacco[J].Plant Molecular Biology,2011,77:17-31.
[10]LI D D,WU Y J,RUAN X M,et al.Expressions of three cotton genes encoding the PIP proteins are regulated in root development and in response to stress[J].Plant Cell Reports,2009,28:291-300.
[11]LI G,TAI F J,ZHENG Y,et al.Two cotton Cys2/His2-type zinc-finger proteins,Gh Di19-1 and Gh Di19-2,are involved in plant response to salt/drought stress and abscisic acid signaling[J].Plant Molecular Biology,2010,74:437-452.
[12]SHI J,ZHANG L,AN H,et al.Gh MPK16,a novel stress-responsive group D MAPK gene from cotton,is involved in disease resistance and drought sensitivity[J].BMC Molecular Biology,2011,12:22.
[13]WANG Q Q,LIU F,CHEN X S,et al.Transcriptome profiling of early developing cotton fiber by deep-sequencing reveals significantly differential expression of genes in a fuzzless/lintless mutant[J].Genomics,2010,96:369-376.
[14]TAO T,ZHAO L,LV Y D,et al.Transcriptome sequencing and differential gene expression analysis of delayed gland morphogenesis in Gossypium australe during seed germination[J].Plos One,2013,8(9):e75323.
[15]HUANG H R,TAN P C,LASCOUX M,et al.Flowering time and transcriptome variation in Capsella bursa-pastoris(Brassicaceae)[J].New Phytologist,2012,194(3):676-689.
[16]WANG X W,LUAN J B,LI J M,et al.De novoccharacterization of a whitefly transcriptome and analysis of its gene expression during development[J].BMC Genomics,2010,11:400.
[17]XIANG L X,HE D,DONG W R,et al.Deep sequencing-based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish[J].BMC Genomics,2010,11:472.
[18]ASMANN Y W,KLEE E W,THOMPSON E A,et al.3’tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer[J].BMC Genomics,2009,10:531.
[19]XUE J,BAO Y Y,Li B L,et al.Transcriptome analysis of the brown planthopper Nilaparvata lugens[J].Plos One,2010,5(12):e14233.
[20]胡根海,喻树迅.利用改良的CTAB法提取棉花叶片总RNA[J].棉花学报,2007,19(1):69-70.
[21]李余良,刘建华,郑锦荣,等.高温胁迫下甜玉米雌穗发育基因差异表达谱分析[J].作物学报,2013,39(2):269-279.
[22]XU P,LIU Z W,FAN X Q,et al.De novo transcriptome sequencing and comparative analysis of differentially expressed genes in Gossypium aridum under salt stress[J].Gene,2013,525:26-34.
[23]LI W,GODZIK A.cd-hit:a fast program for clustering and comparing large sets of protein or nucleotide sequences[J].Bioinformatics,2006,22(13):1658-1659.
[24]ALTSCHUL S F,MADDEN T L,SCHAFFERA A,et al.Gapped BLAST and PSI-BLAST:a new generation of protein database search program[J].Nucleic Acids Research,1997,25:3389-3402.
[25]BOYER J S.Plant productivity and environment[J].Science,1982,218:443-448.
[26]DONSON J,FANG Y,ESPIRITU-SANTO G,et al.Comprehensive gene expression analysis by transcript profiling[J].Plant Molecular Biology,2002,48:75-97.
[27]EVELAND A L,SATOH-NAGASAWA N,GOLDSHMIDT A,et al.Digital gene expression signatures for maize development[J].Plant Physiology,2010,154:1024-1039.
[28]YAO D,ZHANG X,ZHAO X,et al.Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of cotton(Gossypium hirsutum L.)[J].Genomics,2011,98,47-55.
[29]RODRIGUEZ-URIBE L,HIGBIE S M,STEWART J M,et al.Identification of salt responsive genes using comparative microarray analysis inupland cotton(Gossypium hirsutum L.)[J].Plant Science,2011,180:461-469.
[30]WANG G,ZHU Q G,MENG Q W,et al.Transcript profiling during salt stress of young cotton(Gossypium hirsutum)seedlings via Solexa sequencing[J].Acta Physiologiae Plantarum,2012,34(1):107.
[31]CHAUMONT F,BARRIEU F,WOJCIK E,et al.Aquaporins constitute a large and highly divergent protein family in maize[J].Plant Physiology,2001,125:1206-1215.
[32]KATSUHARA M,KOSHIO K,SHIBASAKA M,et al.Overexpression of a barley aquaporin incased the shoot/root ratio and raised salt sensitivity in transgenic rice plants[J].Plant and Cell Physiology,2003,44:1378-1383.
[33]XIANG Y,HUANG Y,XIONG L.Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement[J].Plant Physiology,2007,144:1416-1428.
[34]何亮,李富华,沙莉娜,等.玉米2C型丝氨酸/苏氨酸蛋白磷酸酶(PP2C)活性与耐旱性关系[J].作物学报,2008,34(5):899-903.
[35]MA H L,ZHOU H L,ZHANG H Y,et al.Cloning and expression analysis of an AP2/ERF gene and its responses to phytohormones and abiotic stresses in rice[J].Rice Science,2010,17:1-9.
[36]ZHU Q,ZHANG J,GAO X,et al.The Arabidopsis AP2/ERF transcription factor RAP2.6 participates in ABA,salt and osmotic stress responses[J].Gene,2010,457:1-12.