光皮桦转录组及S-腺苷甲硫氨酸合成酶基因分析
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摘要
转录组测序(transcriptome sequencing)是一种获得基因表达信息的快速、高效方法。为挖掘与光皮桦(Betula luminifera)材质性状相关的基因,本研究以光皮桦茎、叶等组织为材料开展转录组测序、拼接序列的功能注释与分类;采用c DNA末端快速扩增技术(rapid-amplification of c DNA ends,RACE)分离S-腺苷甲硫氨酸合成酶(S-adenosylmethionine synthase,SAMS)基因全长c DNA,再利用DNAStar、MAGA 7.0等生物信息学软件进行序列分析,使用q RT-PCR技术分析SAMS基因的表达模式。结果表明,转录组测序共获得有效数据1.9 Gbp,拼接得到Unigenes序列54 577个。分别比对冗余蛋白质数据库(non-redundant protein database,NR)和Swiss-Prot蛋白质序列数据库(Swiss-Prot protein sequence database),共有65.8%(35920)的Unigenes得到注释信息,包含了24 482个不同序列号的蛋白质。另外,7 954和9 997个Unigenes分别获得了基因本体(Gene Ontology,GO)和直系同源基因簇(Clusters of Orthologous Groups,COG)分类信息。在转录组测序基础上克隆到Bl SAMS1、Bl SAMS2和Bl SAMS3的全长c DNA,其长度分别为1 578 bp、1555 bp和1 266 bp;相应编码蛋白的分子量分别为42.9、43.1和42.7 k D,且皆具典型的SAMS保守结构域。进化树构建显示,Bl SAMS1和长春花(Catharanthus roseus)Cr SAMS2聚在一起;Bl SAMS2和小金海棠(Malus xiaojinensis)Mx SAMS、葡萄(Vitis vinifera)Vv SAMS4聚为一类;Bl SAMS3与木质素合成相关的拟南芥(Arabidopsis thaliana)At SAMS3、海岸松(Pinus pinaster)Pp SAMS1等序列相似,聚在同一分支。Bl SAMS1在叶中的整体表达量较高;Bl SAMS2和Bl SAMS3在茎中的表达量随着木质化程度的增加而升高,且在木质部优势表达。因此,推测Bl SAMS3通过参与光皮桦木质素的合成在木材形成中发挥作用。光皮桦的转录组信息和Bl SAMSs序列及表达分析为进一步研究木材形成的分子机制提供了基础资料。
Transcriptome sequencing is a rapid and efficient way to obtain gene expression information.Betula luminifera is one of the superior woody species in China. In order to excavate the genes involved in its wood formation, the transcriptome sequencing, sequencing data assembly, and functional annotation and classification of the sequences were conducted by using the stems and leaves as materials in this study. Then the full-length c DNAs of S-adenosylmethionine synthase(SAMS) gene were isolated by RACE technique.The sequence feature and phylogenetic tree were analyzed by using bioinformatics software, such as DNAStar, MAGA7.0, and the expression pattern was detected by fluorescence quantitative RCR. The results showed that a total of 1.9 Gbp sequencing data was obtained, and were assembled into 54 577 Unigenes. By BLAST comparison against the NR(non-redundant protein database) and the Swiss-Prot protein sequence database, 65.8%(35 920) of Unigenes were annotated, which corresponded to 24 482 Unique protein accessions. Among these annotated Unigenes, 7 954 and 9 997 were assigned to Gene Ontology(GO) classes and Clusters of Orthologous Groups(COG), respectively. Based on transcriptomes sequencing, Bl SAMS1,Bl SAMS2 and Bl SAMS3 were cloned, with the c DNA length of 1 578 bp, 1 555 bp and 1 266 bp, respectively.The molecular weights of the corresponding encoded proteins were 42.9, 43.1 and 42.7 k D, each of which contained typical conservative domains of SAMS protein. Phylogenetic tree showed that Bl SAMS1 was grouped with Catharanthus roseus SAMS2, and Bl SAMS2, Malus xiaojinensis SAMS, and Vitis vinifera SAMS4 were in the same clade. Bl SAMS3 was clustered into a clade with Arabidopsis thaliana SAMS3 and Pinus pinaster SAMS1, which were involved in lignin biosynthesis. The expression levels of Bl SAMS1 were relatively high in leaf samples. The Bl SAMS2 and Bl SAMS3 were predominantly expressed in stem and xylem,and their expression levels in stem increased as the lignification progressed. It could be speculated that the Bl SAMS3 played an important role in lignin biosynthesis of B. luminifera wood. Transcriptome information and the Bl SAMSs expression patterns could lay a foundation for further dissection on the molecular mechanism of B. luminifera wood formation.
Transcriptome sequencing is a rapid and efficient way to obtain gene expression information.Betula luminifera is one of the superior woody species in China. In order to excavate the genes involved in its wood formation, the transcriptome sequencing, sequencing data assembly, and functional annotation and classification of the sequences were conducted by using the stems and leaves as materials in this study. Then the full-length c DNAs of S-adenosylmethionine synthase(SAMS) gene were isolated by RACE technique.The sequence feature and phylogenetic tree were analyzed by using bioinformatics software, such as DNAStar, MAGA7.0, and the expression pattern was detected by fluorescence quantitative RCR. The results showed that a total of 1.9 Gbp sequencing data was obtained, and were assembled into 54 577 Unigenes. By BLAST comparison against the NR(non-redundant protein database) and the Swiss-Prot protein sequence database, 65.8%(35 920) of Unigenes were annotated, which corresponded to 24 482 Unique protein accessions. Among these annotated Unigenes, 7 954 and 9 997 were assigned to Gene Ontology(GO) classes and Clusters of Orthologous Groups(COG), respectively. Based on transcriptomes sequencing, Bl SAMS1,Bl SAMS2 and Bl SAMS3 were cloned, with the c DNA length of 1 578 bp, 1 555 bp and 1 266 bp, respectively.The molecular weights of the corresponding encoded proteins were 42.9, 43.1 and 42.7 k D, each of which contained typical conservative domains of SAMS protein. Phylogenetic tree showed that Bl SAMS1 was grouped with Catharanthus roseus SAMS2, and Bl SAMS2, Malus xiaojinensis SAMS, and Vitis vinifera SAMS4 were in the same clade. Bl SAMS3 was clustered into a clade with Arabidopsis thaliana SAMS3 and Pinus pinaster SAMS1, which were involved in lignin biosynthesis. The expression levels of Bl SAMS1 were relatively high in leaf samples. The Bl SAMS2 and Bl SAMS3 were predominantly expressed in stem and xylem,and their expression levels in stem increased as the lignification progressed. It could be speculated that the Bl SAMS3 played an important role in lignin biosynthesis of B. luminifera wood. Transcriptome information and the Bl SAMSs expression patterns could lay a foundation for further dissection on the molecular mechanism of B. luminifera wood formation.
引文
黄华宏.2012.基于转录组测序的光皮桦应拉木形成分子机制研究[D].浙江大学,导师:朱睦元,pp.82-98(Huang H H.2012.Studies on molecular mechanism of tension wood formation in Betula luminifera using RNA-Seq[D].Zhejiang University,Supervisor:Zhu M Y,pp.82-98.)
梅利那,范付华,崔博文,等.2017.基于马尾松转录组的SSR分子标记开发及种质鉴定[J].农业生物技术学报25(06):991-1002.(Mei L N,Fang F H,Cui B W,et al2017.Development of SSR molecular markers based on transcriptome sequences and germplasm identification in masson pine(Pinus massoniana)[J].Journal of Agricultural Biotechnology,25(06):991-1002.)
易咏梅,罗世家.1998.光皮桦苗期生长特性的初步研究[J]湖北民族学院学报16(6):16-19.(Yi L M,Luo S J1998.A preliminary study on the growth characteristics of Betula luminifera in seedling stage[J].Journal of Hubei Institute for Nationalities,16(6):16-19.)
余涛,支立峰,彭论,等.2004.烟草中一条新S-腺苷甲硫氨酸合成酶基因的克隆及表达分析[J].武汉植物学研究,22(4):277-283.(Yu T,Zhi L F,Peng L,et al.2004Cloning and characterization of a novel S-adenosyl-lmethionine synthase gene in Tobacco[J].Journal of Wuhan Botanical Research,22(4):277-283.)
朱晶莹,王寒玉,张晏萌,等.2011.玉米S-腺苷甲硫氨酸合成酶基因家族成员在盐胁迫条件下的差异表达[J].核农学报,25(3):427-431.(Zhu J Y,Wang H Y,Zhang YM,et al.2011.Differential expression of maize S-adenosylmethionine synthetase gene family under salt stress[J].Journal of Nuclear Agriculture,25(3):427-431.)
朱妍婕,孔瑾,王忆,等.2009.小金海棠S-腺苷甲硫氨酸合成酶基因(Mx SAMS)的克隆和表达分析[J].农业生物技术学报,17(1):121-125.(Zhu Y J,Kong J,Wang Y,et al.2009.Cloning and expression analysis of S-adenosylmethionine synthetase Gene(Mx SAMS)from Malus hupehensis[J].Journal of Agriculture Biotechnology,17(1):121-125.)
Bai Y,Dougherty L,Xu K.2014.Towards an improved apple reference transcriptome using RNA-seq[J].Molecular genetics and genomics,289(3):427-438.
Belbahri L,Chevalier L,Bensaddek L,et al.2000.Different expression of an S-adenosylmethionine synthetase gene in transgenic tobacco callus modifies alkaloid biosynthesis[J].Biotechnology Bioengineering,69(1):11-20.
Breusegem F V,Dekeyser R,Gielen J,et al.1994.Characterization of a S-adenosylmethionine synthetase gene in rice[J].Plant Physiology,105(4):1463-1464.
Brown G R,Bassoni D L,Gill G P,et al.2003.Identification of quantitative trait loci influencing wood property traits in loblolly pine(Pinus taeda L.).III.QTL verification and candidate gene mapping[J].Genetics.164(4):1537-1546.
Chan K L,New D,Ghandhi S,et al.2002.Transcript levels of the eukaryotic translation initiation factor 5A gene peak at early G1 phase of the cell cycle in the dinoflagellate Crypthecodinium cohnii[J].Applied&Environmental Microbiology,68(5):2278-2284.
Chang L,Chen J J,Xiao Y M,et al.2011.De novo characterization of Lycoris sprengeri transcriptome using illumina GA ii[J].African Journal of Biotechnology,10(57):12147-12155.
Chen J,Chen B,Zhang D.2015.Transcript profiling of Populus tomentosa genes in normal,tension,and opposite wood by RNA-seq[J].BMC Genomics,16(1):164-177.
Constantinos P,Jae-Heung K,Jaemo Y,et al.2005.Transcriptome profiling of vertical stem segments provides insights into the genetic regulation of secondary growth in hybrid aspen trees[J].2005,46(8):1213-1225.
Espartero J,Pintortoro J A,Pardo J M.1994.Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress[J].Plant Molecular Biology,25(2):217-227.
Huang H H,Jiang C,Tong Z K,et al 2014.Eight distinct cellulose synthase catalytic subunit genes from Betula luminifera are associated with primary and secondary cell wall biosynthesis[J].Cellulose,21(4),2183-2198.
Gong W,Qi P,Du J,et al.2014.Transcriptome analysis of shade-induced inhibition on leaf size in relay intercropped soybean[J].Plo S one,9(6):e98465.
Kirst M,Myburg A A,De León J P,et al.2004.Coordinated genetic regulation of growth and lignin revealed by quantitative trait locus analysis of c DNA microarray data in an interspecific backcross of eucalyptus[J].Plant Physiology,135(4):2368-2378.
Lee B R,Kim K Y,Jung W J,et al.2007.Peroxidases and lignification in relation to the intensity of water-deficit stress in white clover(Trifolium repens L.)[J].58(6):1271-1279.
Li R,Zhu H,Ruan J,et al.2010.De novo assembly of human genomes with massively parallel short read sequencing[J].Genome Research,20(2):265-272.
Lindroth A M,Saarikoski P,Flygh G,et al.2001.Two S-adenosylmethionine synthetase-encoding genes differentially expressed during adventitious root development in Pinus contorta[J].Plant Molecular Biology,46(3):335-346.
Meng H,Campbell W H.1998.Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development[J].38(4):513-520.
Peleman J,Boerjan W,Engler G,et al.1989.Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase[J].Plant Cell,1(1):81-93.
Qiu Z,Wan L,Chen T,et al.2013.The regulation of cambial activity in Chinese fir(Cunninghamia lanceolata)involves extensive transcriptome remodeling[J].New Phytologist,199(3):708-719.
Sánchez-Aguayo I,Rodríguez-Galán J M,García R,et al.2004.Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants[J].Planta,220(2):278-285.
Shen B,Li C,Tarczynski M C.2002.High free-methionine and decreased lignin content result from a mutation inthe Arabidopsis S-adenosyl-L-methionine synthetase 3gene[J].The Plant Journal:for cell and molecular biology,29(3):371-380.
Shi C Y,Hua Y,Wei C L,et al.2011.Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compound[J].BMC Genomics,12(1):131-150.
Tabor C W,Tabor H.1984.Methionine adenosyltransferase(S-adenosylmethionine synthetase)and S-adenosylmethionine decarboxylase[J].Advances in Enzymology&Related Areas of Molecular Biology,56:251-282.
Tabuchi T,Kawaguchi Y,Azuma T,et al.2005.Similar regulation patterns of choline monooxygenase,phosphoet hanolamine N-methylt ransferase and S-adenosyl-L-methionine synthetase in leaves of the halophyte Atriplex nummularial[J].Plant&Cell Physiology,46(46):505-513.
Tamura K,Dudley J,Nei M,et al.2007.MEGA4:Molecular evolutionary genetics analysis(MEGA)software version 4.0[J].Molecular Biology&Evolution,24(24):1596-1599.
Thavamanikumar S,Southerton S,Thumma B.2014.RNA-Seq using two populations reveals genes and alleles controlling wood traits and growth in Eucalyptus nitens[J].Plo S one.9(6):e101104.
Tuskan G A,Difazio S,Jansson S,et al.2006.The genome of black cottonwood,Populus trichocarpa(Torr.&Gray)[J].Science,313(5793):1596-1604.
Villalobos D P,Díaz-Moreno S M,Said E S S,et al.2012.Reprogramming of gene expression during compression wood formation in pine:coordinated modulation of S-adenosylmethionine,lignin and lignan related genes[J].BMC Plant Biology,12(1):1-17.
Yang S,Hoffman N.1984.Ethylene biosynthesis and its regulation in higher plants[J].Annual Review Plant Physiology,35(1):155-189.
Yang H,Mao Y X,Yang G P,et al.2011.Profiling of the transcriptome of Porphyra yezoensis with solexa sequencing technology[J].Science Bulletin,56(20):2119-2130.
梅利那,范付华,崔博文,等.2017.基于马尾松转录组的SSR分子标记开发及种质鉴定[J].农业生物技术学报25(06):991-1002.(Mei L N,Fang F H,Cui B W,et al2017.Development of SSR molecular markers based on transcriptome sequences and germplasm identification in masson pine(Pinus massoniana)[J].Journal of Agricultural Biotechnology,25(06):991-1002.)
易咏梅,罗世家.1998.光皮桦苗期生长特性的初步研究[J]湖北民族学院学报16(6):16-19.(Yi L M,Luo S J1998.A preliminary study on the growth characteristics of Betula luminifera in seedling stage[J].Journal of Hubei Institute for Nationalities,16(6):16-19.)
余涛,支立峰,彭论,等.2004.烟草中一条新S-腺苷甲硫氨酸合成酶基因的克隆及表达分析[J].武汉植物学研究,22(4):277-283.(Yu T,Zhi L F,Peng L,et al.2004Cloning and characterization of a novel S-adenosyl-lmethionine synthase gene in Tobacco[J].Journal of Wuhan Botanical Research,22(4):277-283.)
朱晶莹,王寒玉,张晏萌,等.2011.玉米S-腺苷甲硫氨酸合成酶基因家族成员在盐胁迫条件下的差异表达[J].核农学报,25(3):427-431.(Zhu J Y,Wang H Y,Zhang YM,et al.2011.Differential expression of maize S-adenosylmethionine synthetase gene family under salt stress[J].Journal of Nuclear Agriculture,25(3):427-431.)
朱妍婕,孔瑾,王忆,等.2009.小金海棠S-腺苷甲硫氨酸合成酶基因(Mx SAMS)的克隆和表达分析[J].农业生物技术学报,17(1):121-125.(Zhu Y J,Kong J,Wang Y,et al.2009.Cloning and expression analysis of S-adenosylmethionine synthetase Gene(Mx SAMS)from Malus hupehensis[J].Journal of Agriculture Biotechnology,17(1):121-125.)
Bai Y,Dougherty L,Xu K.2014.Towards an improved apple reference transcriptome using RNA-seq[J].Molecular genetics and genomics,289(3):427-438.
Belbahri L,Chevalier L,Bensaddek L,et al.2000.Different expression of an S-adenosylmethionine synthetase gene in transgenic tobacco callus modifies alkaloid biosynthesis[J].Biotechnology Bioengineering,69(1):11-20.
Breusegem F V,Dekeyser R,Gielen J,et al.1994.Characterization of a S-adenosylmethionine synthetase gene in rice[J].Plant Physiology,105(4):1463-1464.
Brown G R,Bassoni D L,Gill G P,et al.2003.Identification of quantitative trait loci influencing wood property traits in loblolly pine(Pinus taeda L.).III.QTL verification and candidate gene mapping[J].Genetics.164(4):1537-1546.
Chan K L,New D,Ghandhi S,et al.2002.Transcript levels of the eukaryotic translation initiation factor 5A gene peak at early G1 phase of the cell cycle in the dinoflagellate Crypthecodinium cohnii[J].Applied&Environmental Microbiology,68(5):2278-2284.
Chang L,Chen J J,Xiao Y M,et al.2011.De novo characterization of Lycoris sprengeri transcriptome using illumina GA ii[J].African Journal of Biotechnology,10(57):12147-12155.
Chen J,Chen B,Zhang D.2015.Transcript profiling of Populus tomentosa genes in normal,tension,and opposite wood by RNA-seq[J].BMC Genomics,16(1):164-177.
Constantinos P,Jae-Heung K,Jaemo Y,et al.2005.Transcriptome profiling of vertical stem segments provides insights into the genetic regulation of secondary growth in hybrid aspen trees[J].2005,46(8):1213-1225.
Espartero J,Pintortoro J A,Pardo J M.1994.Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress[J].Plant Molecular Biology,25(2):217-227.
Huang H H,Jiang C,Tong Z K,et al 2014.Eight distinct cellulose synthase catalytic subunit genes from Betula luminifera are associated with primary and secondary cell wall biosynthesis[J].Cellulose,21(4),2183-2198.
Gong W,Qi P,Du J,et al.2014.Transcriptome analysis of shade-induced inhibition on leaf size in relay intercropped soybean[J].Plo S one,9(6):e98465.
Kirst M,Myburg A A,De León J P,et al.2004.Coordinated genetic regulation of growth and lignin revealed by quantitative trait locus analysis of c DNA microarray data in an interspecific backcross of eucalyptus[J].Plant Physiology,135(4):2368-2378.
Lee B R,Kim K Y,Jung W J,et al.2007.Peroxidases and lignification in relation to the intensity of water-deficit stress in white clover(Trifolium repens L.)[J].58(6):1271-1279.
Li R,Zhu H,Ruan J,et al.2010.De novo assembly of human genomes with massively parallel short read sequencing[J].Genome Research,20(2):265-272.
Lindroth A M,Saarikoski P,Flygh G,et al.2001.Two S-adenosylmethionine synthetase-encoding genes differentially expressed during adventitious root development in Pinus contorta[J].Plant Molecular Biology,46(3):335-346.
Meng H,Campbell W H.1998.Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development[J].38(4):513-520.
Peleman J,Boerjan W,Engler G,et al.1989.Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase[J].Plant Cell,1(1):81-93.
Qiu Z,Wan L,Chen T,et al.2013.The regulation of cambial activity in Chinese fir(Cunninghamia lanceolata)involves extensive transcriptome remodeling[J].New Phytologist,199(3):708-719.
Sánchez-Aguayo I,Rodríguez-Galán J M,García R,et al.2004.Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants[J].Planta,220(2):278-285.
Shen B,Li C,Tarczynski M C.2002.High free-methionine and decreased lignin content result from a mutation inthe Arabidopsis S-adenosyl-L-methionine synthetase 3gene[J].The Plant Journal:for cell and molecular biology,29(3):371-380.
Shi C Y,Hua Y,Wei C L,et al.2011.Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compound[J].BMC Genomics,12(1):131-150.
Tabor C W,Tabor H.1984.Methionine adenosyltransferase(S-adenosylmethionine synthetase)and S-adenosylmethionine decarboxylase[J].Advances in Enzymology&Related Areas of Molecular Biology,56:251-282.
Tabuchi T,Kawaguchi Y,Azuma T,et al.2005.Similar regulation patterns of choline monooxygenase,phosphoet hanolamine N-methylt ransferase and S-adenosyl-L-methionine synthetase in leaves of the halophyte Atriplex nummularial[J].Plant&Cell Physiology,46(46):505-513.
Tamura K,Dudley J,Nei M,et al.2007.MEGA4:Molecular evolutionary genetics analysis(MEGA)software version 4.0[J].Molecular Biology&Evolution,24(24):1596-1599.
Thavamanikumar S,Southerton S,Thumma B.2014.RNA-Seq using two populations reveals genes and alleles controlling wood traits and growth in Eucalyptus nitens[J].Plo S one.9(6):e101104.
Tuskan G A,Difazio S,Jansson S,et al.2006.The genome of black cottonwood,Populus trichocarpa(Torr.&Gray)[J].Science,313(5793):1596-1604.
Villalobos D P,Díaz-Moreno S M,Said E S S,et al.2012.Reprogramming of gene expression during compression wood formation in pine:coordinated modulation of S-adenosylmethionine,lignin and lignan related genes[J].BMC Plant Biology,12(1):1-17.
Yang S,Hoffman N.1984.Ethylene biosynthesis and its regulation in higher plants[J].Annual Review Plant Physiology,35(1):155-189.
Yang H,Mao Y X,Yang G P,et al.2011.Profiling of the transcriptome of Porphyra yezoensis with solexa sequencing technology[J].Science Bulletin,56(20):2119-2130.