不同盐度胁迫下杜氏盐藻全转录组测序及注释
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摘要
【目的】通过对杜氏盐藻的转录组进行测序和基因功能分析,阐明不同浓度盐胁迫对杜氏盐藻生长发育以及不同信号途径的影响。【方法】分别获取9%NaCl浓度和24%NaCl浓度培养下的杜氏盐藻转录组并通过Illumina平台进行测序。将所得的序列进行拼接、去冗余处理。【结果】获得40682个unigenes,其中注释到NR数据库的10905个,注释到NT数据库的2768个,注释到SWISS-PROT数据库的7261个,注释到COG/KOG数据库的6499个。受到高盐胁迫的杜氏盐藻细胞相比低盐环境下,有717个基因表达上调,1012个基因表达下调。进一步对60个显著差异基因进行了功能聚类,发现盐胁迫诱导了光合作用途径的基因表达。【结论】杜氏盐藻通过提高光合作用基因表达增强耐盐性。该研究最大范围上挖掘了杜氏盐藻在高盐和低盐环境的基因转录水平,为深入揭示杜氏盐藻盐胁迫下基因差异表达提供了平台,并为进一步研究杜氏盐藻耐盐机理提供理论依据。
[Objective] Through sequencing and genetic function analysis of the transcripts of Dunaliella salina, we studied its response to salt stress. [Methods] The transcriptome of Dunaliella salina under 9% and 24% NaCl concentration was obtained and sequenced by Illumina platform. All the unigenes were finally obtained by sequence splicing, redundancy removing based on the sequence clustering software. Functional notation and cluster analysis of unigenes were performed by comparing the unigenes to the database. [Results] A total of 40682 unigenes were obtained, including 10905 in the NR database, 2768 in the NT database, 7261 in the Swiss-prot database, and 6499 in the COG/KOG database. By comparing cells grown under hyper-and hyposaline conditions,717 genes(41.46%) increased expression and 1012 genes(58.5%) decreased expression. Through the data integration analysis, 60 different expression genes of Dunaliella salina under salt stress were excavated.[Conclusion] Salinity stress up-regulated key genes in photosynthesis. Our findings provide reference for dissecting molecular mechanisms of salinity tolerance in algae and higher plants.
[Objective] Through sequencing and genetic function analysis of the transcripts of Dunaliella salina, we studied its response to salt stress. [Methods] The transcriptome of Dunaliella salina under 9% and 24% NaCl concentration was obtained and sequenced by Illumina platform. All the unigenes were finally obtained by sequence splicing, redundancy removing based on the sequence clustering software. Functional notation and cluster analysis of unigenes were performed by comparing the unigenes to the database. [Results] A total of 40682 unigenes were obtained, including 10905 in the NR database, 2768 in the NT database, 7261 in the Swiss-prot database, and 6499 in the COG/KOG database. By comparing cells grown under hyper-and hyposaline conditions,717 genes(41.46%) increased expression and 1012 genes(58.5%) decreased expression. Through the data integration analysis, 60 different expression genes of Dunaliella salina under salt stress were excavated.[Conclusion] Salinity stress up-regulated key genes in photosynthesis. Our findings provide reference for dissecting molecular mechanisms of salinity tolerance in algae and higher plants.
引文
[1]Geng DG,Han Y,Wang YQ,Li WB,Sun YR.Progress in studies on salt-resistant mechanism and prospects of gene engineering of Dunaliella salina.Chinese Bulletin of Botany,2002,19(3):290-295.(in Chinese)耿德贵,韩燕,王义琴,李文彬,孙勇如.杜氏盐藻的耐盐机制研究进展和基因工程研究的展望.植物学通报,2002,19(3):290-295.
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[6]Shu SH,Chen B,Zhou MC,Zhao XM,Xia HY,Wang M.De novo sequencing and transcriptome analysis of Wolfiporia cocos to reveal genes related to biosynthesis of triterpenoids.PLoS One,2013,8(8):e71350.
[7]Nagalakshmi U,Wang Z,Waern K,Shou C,Raha D,Gerstein M,Snyder M.The transcriptional landscape of the yeast genome defined by RNA sequencing.Science,2008,320(5881):1344-1349.
[8]Lister R,O’Malley RC,Tonti-Filippini J,Gregory BD,Berry CC,Millar AH,Ecker JR.Highly integrated single-base resolution maps of the epigenome in Arabidopsis.Cell,2008,133(3):523-536.
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[10]Morin RD,Bainbridge M,Fejes A,Hirst M,Krzywinski M,Pugh TJ,McDonald H,Varhol R,Jones SJM,Marra MA.Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing.Biotechniques,2008,45(1):81-94.
[11]Xu P,Liu ZW,Fan XQ,Gao J,Zhang X,Zhang XG,Shen XL.De novo transcriptome sequencing and comparative analysis of differentially expressed genes in Gossypium aridum under salt stress.Gene,2013,525(1):26-34.
[12]Wang XC,Zhao QY,Ma CL,Zhang ZH,Gao HL,Kong YM,Yue C,Hao XY,Chen L,Ma JQ,Li X,Yang YJ.Global transcriptome profiles of Camellia sinensis during cold acclimation.BMC Genomics,2013,14:415.
[13]Xu WR,Li RM,Zhang NB,Ma FL,Jiao YT,Wang ZP.Transcriptome profiling of Vitis amurensis,an extremely cold-tolerant Chinese wild Vitis species,reveals candidate genes and events that potentially connected to cold stress.Plant Molecular Biology,2014,86(4/5):527-541.
[14]Liu H,Ouyang B,Zhang JH,Wang TT,Li HX,Zhang YY,Yu CY,Ye ZB.Differential modulation of photosynthesis,signaling,and transcriptional regulation between tolerant and sensitive tomato genotypes under cold stress.PLoS One,2012,7(11):e50785.
[15]Chen HY,Chen XL,Chen D,Li JF,Zhang Y,Wang A.Acomparison of the low temperature transcriptomes of two tomato genotypes that differ in freezing tolerance:Solanum lycopersicum and Solanum habrochaites.BMC Plant Biology,2015,15:132.
[16]Wang JM,Yang Y,Liu XH,Li J,Zhang Q,Gu JH,Lu YM.Transcriptome profiling of the cold response and signaling pathways in Lilium lancifolium.BMC Genomics,2014,15:203.
[17]Yate SA,Swain MT,Hegarty MJ,Chernukin I,Lowe M,Allison GG,Ruttink T,Abberton MT,Jenkins G,Sk?t L.De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response,gene discovery and marker identification.BMC Genomics,2014,15:453.
[18]Liu JL,Hong L.Energy metabolic pathways in Dunaleilla salina using the transcriptome data.Geomatics and Information Science of Wuhan University,2018,64(1):84-94.(in Chinese)刘军立,红凌.基于转录组数据分析杜氏盐藻能量代谢途径.武汉大学学报,2018,64(1):84-94.
[19]Zhu LQ,Li QH,Zhang YT,Zhu XZ,Guan FX,Xue LX.Transcriptome sequencing and analysis of Dunaliella salina based on RNA-seq.Journal of Zhengzhou University(Medical Sciences),2016,51(3):289-293.(in Chinese)朱立强,李庆华,张彦婷,朱相展,关方霞,薛乐勋.基于RNA-seq的杜氏盐藻全转录组测序与分析.郑州大学学报(医学版),2016,51(3):289-293.
[20]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,2001,25(4):402-408
[21]Zhou L,Meng XH,Liu CS,Yu LJ,Chen XG.Effects of osmotic stress on intracellular glycerol content and enzyme activity in Dunaliella salina.Chinese Bulletin of Botany,2006,23(2):145-151.(in Chinese)周丽,孟祥红,刘成圣,于乐军,陈西广.渗透胁迫对杜氏盐藻胞内甘油含量及相关酶活性影响.植物学通报,2006,23(2):145-151.
[22]Han DM,Chai XJ,Wang YY,Liu SC,Yue WJ.Cloning and expression analysis of DsPLC under salt stress from Dunaliella salina.Journal of Nuclear Agricultural Sciences,2014,28(10):1773-1780.(in Chinese)韩冬梅,柴晓杰,王逸云,刘世才,岳文静.杜氏盐藻磷脂酶C基因DsPLC的克隆及盐胁迫下的表达分析.核农学报,2014,28(10):1773-1780.
[23]Fisher M,Pick U,Zamir A.A salt-induced 60-kilodalton plasma membrane protein plays a potential role in the extreme halotolerance of the alga Dunaliella.Plant Physiology,1994,106(4):1359-1365.
[24]Fisher M,Gokhman I,Pick U,Zamir A.A Salt-resistant plasma membrane carbonic anhydrase is induced by salt in Dunaliella salina.Journal of Biological Chemistry,1996,271(30):17718-17723.
[25]Zhang C,Tang CC,Wang JY,Guo LM,Wang LL,Li WK.Research progress of transcriptomics in plant response to stress.Journal of Biology,2017,34(2):86-90.(in Chinese)张纯,唐承晨,王吉永,郭龙妹,王莉莉,黎万奎.转录组学在植物应答逆境胁迫中的研究进展.生物学杂志,2017,34(2):86-90.
[26]Osmond CB,Foyer CH,Bock G,Wingler A,Lea PJ,Quick WP,Leegood RC.Photorespiration:metabolic pathways and their role in stress protection.Philosophical Transactions of the Royal of London Society.Series B:Biological Sciences,2000,355(1402):1517-1529.
[2]Sadka A,Himmelhoch S,Zamir A.A 150 kilodalton cell surface protein is induced by salt in the halotolerant green alga Dunaliella salina.Plant Physiology,1991,95(3):822-831.
[3]Parida AK,Das AB.Salt tolerance and salinity effects on plants:a review.Ecotoxicology and Environmental Safety,2005,60(3):324-349.
[4]Liu HL,Zheng LM,Liu QQ,Quan FS,Zhang Y.Studies on the transcriptomes of non-model organisms.Hereditas,2013,35(8):955-970.(in Chinese)刘红亮,郑丽明,刘青青,权富生,张涌.非模式生物转录组研究.遗传,2013,35(8):955-970.
[5]Wu Q,Zhang L,Huang ZP,Wang DG,Hu GY.Transcription sequencing and its application on discovering the gene resources of wild soybean.Soybean Science,2013,32(6):845-851.(in Chinese)吴倩,张磊,黄志平,王大刚,胡国玉.转录组测序及其在野生大豆基因资源发掘中的应用.大豆科学,2013,32(6):845-851.
[6]Shu SH,Chen B,Zhou MC,Zhao XM,Xia HY,Wang M.De novo sequencing and transcriptome analysis of Wolfiporia cocos to reveal genes related to biosynthesis of triterpenoids.PLoS One,2013,8(8):e71350.
[7]Nagalakshmi U,Wang Z,Waern K,Shou C,Raha D,Gerstein M,Snyder M.The transcriptional landscape of the yeast genome defined by RNA sequencing.Science,2008,320(5881):1344-1349.
[8]Lister R,O’Malley RC,Tonti-Filippini J,Gregory BD,Berry CC,Millar AH,Ecker JR.Highly integrated single-base resolution maps of the epigenome in Arabidopsis.Cell,2008,133(3):523-536.
[9]Mortazavi A,Williams BA,Mccue K,Schaeffer L,Wold B.Mapping and quantifying mammalian transcriptomes by RNA-Seq.Nature Methods,2008,5(7):621-628.
[10]Morin RD,Bainbridge M,Fejes A,Hirst M,Krzywinski M,Pugh TJ,McDonald H,Varhol R,Jones SJM,Marra MA.Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing.Biotechniques,2008,45(1):81-94.
[11]Xu P,Liu ZW,Fan XQ,Gao J,Zhang X,Zhang XG,Shen XL.De novo transcriptome sequencing and comparative analysis of differentially expressed genes in Gossypium aridum under salt stress.Gene,2013,525(1):26-34.
[12]Wang XC,Zhao QY,Ma CL,Zhang ZH,Gao HL,Kong YM,Yue C,Hao XY,Chen L,Ma JQ,Li X,Yang YJ.Global transcriptome profiles of Camellia sinensis during cold acclimation.BMC Genomics,2013,14:415.
[13]Xu WR,Li RM,Zhang NB,Ma FL,Jiao YT,Wang ZP.Transcriptome profiling of Vitis amurensis,an extremely cold-tolerant Chinese wild Vitis species,reveals candidate genes and events that potentially connected to cold stress.Plant Molecular Biology,2014,86(4/5):527-541.
[14]Liu H,Ouyang B,Zhang JH,Wang TT,Li HX,Zhang YY,Yu CY,Ye ZB.Differential modulation of photosynthesis,signaling,and transcriptional regulation between tolerant and sensitive tomato genotypes under cold stress.PLoS One,2012,7(11):e50785.
[15]Chen HY,Chen XL,Chen D,Li JF,Zhang Y,Wang A.Acomparison of the low temperature transcriptomes of two tomato genotypes that differ in freezing tolerance:Solanum lycopersicum and Solanum habrochaites.BMC Plant Biology,2015,15:132.
[16]Wang JM,Yang Y,Liu XH,Li J,Zhang Q,Gu JH,Lu YM.Transcriptome profiling of the cold response and signaling pathways in Lilium lancifolium.BMC Genomics,2014,15:203.
[17]Yate SA,Swain MT,Hegarty MJ,Chernukin I,Lowe M,Allison GG,Ruttink T,Abberton MT,Jenkins G,Sk?t L.De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response,gene discovery and marker identification.BMC Genomics,2014,15:453.
[18]Liu JL,Hong L.Energy metabolic pathways in Dunaleilla salina using the transcriptome data.Geomatics and Information Science of Wuhan University,2018,64(1):84-94.(in Chinese)刘军立,红凌.基于转录组数据分析杜氏盐藻能量代谢途径.武汉大学学报,2018,64(1):84-94.
[19]Zhu LQ,Li QH,Zhang YT,Zhu XZ,Guan FX,Xue LX.Transcriptome sequencing and analysis of Dunaliella salina based on RNA-seq.Journal of Zhengzhou University(Medical Sciences),2016,51(3):289-293.(in Chinese)朱立强,李庆华,张彦婷,朱相展,关方霞,薛乐勋.基于RNA-seq的杜氏盐藻全转录组测序与分析.郑州大学学报(医学版),2016,51(3):289-293.
[20]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,2001,25(4):402-408
[21]Zhou L,Meng XH,Liu CS,Yu LJ,Chen XG.Effects of osmotic stress on intracellular glycerol content and enzyme activity in Dunaliella salina.Chinese Bulletin of Botany,2006,23(2):145-151.(in Chinese)周丽,孟祥红,刘成圣,于乐军,陈西广.渗透胁迫对杜氏盐藻胞内甘油含量及相关酶活性影响.植物学通报,2006,23(2):145-151.
[22]Han DM,Chai XJ,Wang YY,Liu SC,Yue WJ.Cloning and expression analysis of DsPLC under salt stress from Dunaliella salina.Journal of Nuclear Agricultural Sciences,2014,28(10):1773-1780.(in Chinese)韩冬梅,柴晓杰,王逸云,刘世才,岳文静.杜氏盐藻磷脂酶C基因DsPLC的克隆及盐胁迫下的表达分析.核农学报,2014,28(10):1773-1780.
[23]Fisher M,Pick U,Zamir A.A salt-induced 60-kilodalton plasma membrane protein plays a potential role in the extreme halotolerance of the alga Dunaliella.Plant Physiology,1994,106(4):1359-1365.
[24]Fisher M,Gokhman I,Pick U,Zamir A.A Salt-resistant plasma membrane carbonic anhydrase is induced by salt in Dunaliella salina.Journal of Biological Chemistry,1996,271(30):17718-17723.
[25]Zhang C,Tang CC,Wang JY,Guo LM,Wang LL,Li WK.Research progress of transcriptomics in plant response to stress.Journal of Biology,2017,34(2):86-90.(in Chinese)张纯,唐承晨,王吉永,郭龙妹,王莉莉,黎万奎.转录组学在植物应答逆境胁迫中的研究进展.生物学杂志,2017,34(2):86-90.
[26]Osmond CB,Foyer CH,Bock G,Wingler A,Lea PJ,Quick WP,Leegood RC.Photorespiration:metabolic pathways and their role in stress protection.Philosophical Transactions of the Royal of London Society.Series B:Biological Sciences,2000,355(1402):1517-1529.
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