中华猕猴桃基因组可变剪接事件鉴定及分析
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摘要
可变剪接使一个基因能产生多种m RNA成熟体,极大地增加蛋白多样性.采用中华猕猴桃基因组数据做参考数据,利用中华猕猴桃叶片和果实3个不同发育时期(未成熟、半成熟和成熟期)的转录组数据,从中华猕猴桃基因组(39040个基因)中共鉴定出11651个基因(占总基因数的29%)对应的32180个可变剪接事件.在可变剪接不同类型中,内含子保留类型的发生频率最高,占50%以上;3′可变位点类型频率约为5′端可变类型的2倍.GO富集分析结果表明,可变剪接的基因主要富集于酶调控及核苷酸结合相关功能的GO类别中,而组织特有可变剪接基因功能富集热点与组织的重要功能关联,叶片多为肌动蛋白及微管相关;未成熟果实与双组分信号系统相关;半成熟果实多与磷脂合成过程相关;成熟果实多与信号传递过程相关.另外,55.6%的维生素合成相关基因发生可变剪接事件,显著高于基因组水平的29.6%,暗示着可变剪接参与维生素合成相关基因代谢过程中的重要作用.通过对中华猕猴桃全基因组可变剪接的分析,为解析中华猕猴桃基因组及进一步开展相关分子育种工作提供依据.
Alternative splicing can increase the diversity and complexity of proteome greatly through creating multiple m RNA transcripts from a single gene. RNA-seq data from different organs(leaf, immature fruit, half-ripe fruit and mature fruit) of Actinidia chinensis had been used to identify the alternative splicing events using the A. chinensis genome as reference, and totally 32180 alternative splicing events were discovered from the A. chinensis genome(39040 genes) corresponding to 11651 genes(29% of the total genome). Intron retention events showed the highest frequency(more than 50%) among all the different alternative splicing events. The frequency of events with alternative 3′ splice sites was twice than the alternative 5′ splice sites. GO enrichment analysis result showed that the alternative splicing gene was mainly enriched in enzyme regulation and nucleotide binding related GO categories. The alternative splicing events in different organs of A. chinensis seemed to preferentially occur in genes with important function. The genes of leaf were associated with the function of actin and microtubule. The genes of immature fruit decided the function of two-component signal system. The genes of half-ripe fruit were connected with phospholipid synthesis process. The genes in mature fruit were associated with signal transduction. In addition, 55.6% of vitamin related genes had been found to have alternative splicing changes, which were significantly higher than the average changes of the genome(the average splicing change level was 29.6%). Those explained that alternative splicing events played an important role in the process of vitamin synthesis. In this paper, through the genome-wide analysis of alternative splicing in A. chinensis, a powerful resource for understanding the complex genome of A. chinensis was provided. The result was also useful to molecular breeding in kiwifruit.
Alternative splicing can increase the diversity and complexity of proteome greatly through creating multiple m RNA transcripts from a single gene. RNA-seq data from different organs(leaf, immature fruit, half-ripe fruit and mature fruit) of Actinidia chinensis had been used to identify the alternative splicing events using the A. chinensis genome as reference, and totally 32180 alternative splicing events were discovered from the A. chinensis genome(39040 genes) corresponding to 11651 genes(29% of the total genome). Intron retention events showed the highest frequency(more than 50%) among all the different alternative splicing events. The frequency of events with alternative 3′ splice sites was twice than the alternative 5′ splice sites. GO enrichment analysis result showed that the alternative splicing gene was mainly enriched in enzyme regulation and nucleotide binding related GO categories. The alternative splicing events in different organs of A. chinensis seemed to preferentially occur in genes with important function. The genes of leaf were associated with the function of actin and microtubule. The genes of immature fruit decided the function of two-component signal system. The genes of half-ripe fruit were connected with phospholipid synthesis process. The genes in mature fruit were associated with signal transduction. In addition, 55.6% of vitamin related genes had been found to have alternative splicing changes, which were significantly higher than the average changes of the genome(the average splicing change level was 29.6%). Those explained that alternative splicing events played an important role in the process of vitamin synthesis. In this paper, through the genome-wide analysis of alternative splicing in A. chinensis, a powerful resource for understanding the complex genome of A. chinensis was provided. The result was also useful to molecular breeding in kiwifruit.
引文
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4 张菊明,林佩芳.中华猕猴桃多糖对巨噬细胞-T细胞免疫介质的作用.科技通报,1990,6:284–286
5 宋文瑛,许冠华,张光霁.猕猴桃根多糖对人胃癌SGC-7901细胞增殖、凋亡及p-p38表达的影响.中国中西医结合杂志,2014,34:329 –333
6 Wang B B,Brendel V.Genomewide comparative analysis of alternative splicing in plants.Proc Natl Acad Sci USA,2006,103:7175–7180
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9 Tong C,Wang X,Yu J,et al.Comprehensive analysis of RNA-seq data reveals the complexity of the transcriptome in Brassica rapa.BMC Genomics,2013,14:689
10 Reddy A S.Alternative splicing of pre-messenger RNAs in plants in the genomic era.Annu Rev Plant Biol,2007,58:267–294
11 Zhang G,Guo G,Hu X,et al.Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome.Genome Res,2010,20:646–654
12 Reddy A S,Marquez Y,Kalyna M,et al.Complexity of the alternative splicing landscape in plants.Plant Cell,2013,25:3657–3683
13 Zhang X N,Mount S M.Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.Plant Physiol,2009,150:1450–1458
14 Wang Q,Silver P A.Genome-wide RNAi screen discovers functional coupling of alternative splicing and cell cycle control to apoptosis regulation.Cell Cycle,2010,9:4419–4421
15 Gu L,Guo R.Genome-wide detection and analysis of alternative splicing for nucleotide binding site-leucine-rich repeats sequences in rice.J genet genomics,2007,34:247–257
16 Leviatan N,Alkan N,Leshkowitz D,et al.Genome-wide survey of cold stress regulated alternative splicing in Arabidopsis thaliana with tiling microarray.PLo S One,2013,8:e66511
17 Staiger D,Brown J W.Alternative splicing at the intersection of biological timing,development,and stress responses.Plant Cell,2013,25 :3640–3656
18 Quesada V,Macknight R,Dean C,et al.Autoregulation of FCA pre-m RNA processing controls Arabidopsis flowering time.EMBO J,2003,22:3142–3152
19 Ding F,Cui P,Wang Z Y,et al.Genome-wide analysis of alternative splicing of pre-m RNA under salt stress in Arabidopsis.BMC Genomics,2014,15:431
20 Wang E T,Sandberg R,Luo S,et al.Alternative isoform regulation in human tissue transcriptomes.Nature,2008,456:470–476
21 Filichkin S A,Priest H D,Givan S A,et al.Genome-wide mapping of alternative splicing in Arabidopsis thaliana.Genome Res,2010,20:45 –58
22 Goldberg D H,Victor J D,Gardner E P,et al.Spike train analysis toolkit:enabling wider application of information-theoretic techniques to neurophysiology.Neuroinformatics,2009,7:165–178
23 Patel R K,Jain M.NGS QC Toolkit:a toolkit for quality control of next generation sequencing data.PLo S One,2012,7:e30619
24 Trapnell C,Pachter L,Salzberg S L.Top Hat:discovering splice junctions with RNA-Seq.Bioinformatics,2009,25:1105–1111
25 Quevillon E,Silventoinen V,Pillai S,et al.Inter Pro Scan:protein domains identifier.Nucleic Acids Res,2005,33:W116–W120
26 Trapnell C,Roberts A,Goff L,et al.Differential gene and transcript expression analysis of RNA-seq experiments with Top Hat and Cufflinks.Nat Protoc,2012,7:562–578
27 Rabbitts T H.Evidence for splicing of interrupted immunoglobulin variable and constant region sequences in nuclear RNA.Nature,1978,275 :291–296
28 Black D L.Mechanisms of alternative pre-messenger RNA splicing.Annu Rev Biochem,2003,72:291–336
29 Stamm S,Ben-Ari S,Rafalska I,et al.Function of alternative splicing.Gene,2005,344:1–20
30 Lareau L F,Green R E,Bhatnagar R S,et al.The evolving roles of alternative splicing.Curr Opin Struc Biol,2004,14:273–282
31 Xu Q,Modrek B,Lee C.Genome-wide detection of tissue-specific alternative splicing in the human transcriptome.Nucleic Acids Res,2002,30:3754–3766
32 Transcriptome and genome conservation of alternative splicing events in humans and mice.In:Sugnet C W,Kent W J,Haussler D,eds.Proc.9th Pacific Symposium on Biocomputing.Hawaii.2004,Singapore:World Scientific,2004.66–77
33 Chacko E,Ranganathan S.Genome-wide analysis of alternative splicing in cow:implications in bovine as a model for human diseases.BMC Genomics,2009,10:S11
34 Babenko V N,Aitnazarov R B,Goncharov F A,et al.Alternative splicing landscape of the Drosophila melanogaster genome.Russ J Genet,2010,46:1036–1038
35 Labadorf A,Link A,Rogers M F,et al.Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii.BMC Genomics,2010,11:114
36 Ramani A K,Calarco J A,Pan Q,et al.Genome-wide analysis of alternative splicing in Caenorhabditis elegans.Genome Res,2011,21:342 –348
37 Sablok G,Gupta P K,Baek J M,et al.Genome-wide survey of alternative splicing in the grass Brachypodium distachyon:a emerging model biosystem for plant functional genomics.Biotechnol Lett,2011,33:629–636
38 Bao H,Li E Y,Mansfield S D,et al.The developing xylem transcriptome and genome-wide analysis of alternative splicing in Populus trichocarpa(black cottonwood)populations.BMC Genomics,2013,14:359
39 Panahi B,Abbaszadeh B,Taghizadeghan M,et al.Genome-wide survey of alternative splicing in Sorghum Bicolor.Physiol Mol Biol Plants,2014,20:323–329
40 Wu H P,Su Y S,Chen H C,et al.Genome-wide analysis of light-regulated alternative splicing mediated by photoreceptors in Physcomitrella patens.Genome Biol,2014,15:R10
41 Shen Y,Zhou Z,Wang Z,et al.Global dissection of alternative splicing in paleopolyploid soybean.Plant Cell,2014,26:996–1008
42 Li P,Ponnala L,Gandotra N,et al.The developmental dynamics of the maize leaf transcriptome.Nat Genet,2010,42:1060–1067
43 Marquez Y,Brown J W,Simpson C,et al.Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis.Genome Res,2012,22:1184–1195
44 Pan Q,Shai O,Lee L J,et al.Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.Nat Genet,2008,40:1413–1415
45 Walters B,Lum G,Sablok G,et al.Genome-wide landscape of alternative splicing events in Brachypodium distachyon.DNA Res,2013,20 :163–171
46 Baek J M,Han P,Iandolino A,et al.Characterization and comparison of intron structure and alternative splicing between Medicago truncatula,Populus trichocarpa,Arabidopsis and rice.Plant Mol Biol,2008,67:499–510
47 Staiger C J.Signaling to the actin cytoskeleton in plants.Annu Rev Plant Physiol Plant Mol Biol,2000,51:257–288
48 Kadota A,Wada M.Photoorientation of chloroplasts in protonemal cells of the fern Adiantum as analyzed by use of a video-tracking system.Bot Mag,1992,105:265–279
49 Wada M,Kagawa T,Sato Y.Chloroplast movement.Annu Rev Plant Biol,2003,54:455–468
50 Stock A M,Robinson V L,Goudreau P N.Two-component signal transduction.Annu Rev Biochem,2000,69:183–215
51 吴家森,管剑峰.秦美猕猴桃果实生育及营养量变的若干特点.浙江林学院学报,2002,19:244–246
52 安华明,樊卫国,刘进平.生育期猕猴桃果实中营养元素积累规律研究.种子,2003,4:24–25
53 陈昆松.ABA和IAA对猕猴桃果实成熟进程的调控.园艺学报,1999,26:81–86
54 张玉,陈昆松,张上隆,等.猕猴桃果实采后成熟过程中糖代谢及其调节.植物生理与分子生物学学报,2004,30:317–324
55 涂正顺,李华,王华,等.猕猴桃果实采后香气成分的变化.园艺学报,2001,28:512–516
56 殷学仁,张波,李鲜,等.乙烯信号转导与果实成熟衰老的研究进展.园艺学报,2009,36:133–140
2 Cheng C H,Seal A G,Boldingh H L,et al.Inheritance of taste characters and fruit size and number in a diploid Actinidia chinensis(kiwifruit)population.Euphytica,2004,138:185–195
3 Skinner M A,Loh J M S,Hunter D C,et al.Gold kiwifruit(Actinidia chinensis‘Hort16A’)for immune support.Proc Nutr Soc,2011,70:276 –280
4 张菊明,林佩芳.中华猕猴桃多糖对巨噬细胞-T细胞免疫介质的作用.科技通报,1990,6:284–286
5 宋文瑛,许冠华,张光霁.猕猴桃根多糖对人胃癌SGC-7901细胞增殖、凋亡及p-p38表达的影响.中国中西医结合杂志,2014,34:329 –333
6 Wang B B,Brendel V.Genomewide comparative analysis of alternative splicing in plants.Proc Natl Acad Sci USA,2006,103:7175–7180
7 Huang S,Ding J,Deng D,et al.Draft genome of the kiwifruit Actinidia chinensis.Nat Commun,2013,4:2640
8 Berget S M,Moore C,Sharp P A.Spliced segments at the 5′terminus of adenovirus 2 late m RNA.Proc Natl Acad Sci USA,1977,74:3171–3175
9 Tong C,Wang X,Yu J,et al.Comprehensive analysis of RNA-seq data reveals the complexity of the transcriptome in Brassica rapa.BMC Genomics,2013,14:689
10 Reddy A S.Alternative splicing of pre-messenger RNAs in plants in the genomic era.Annu Rev Plant Biol,2007,58:267–294
11 Zhang G,Guo G,Hu X,et al.Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome.Genome Res,2010,20:646–654
12 Reddy A S,Marquez Y,Kalyna M,et al.Complexity of the alternative splicing landscape in plants.Plant Cell,2013,25:3657–3683
13 Zhang X N,Mount S M.Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.Plant Physiol,2009,150:1450–1458
14 Wang Q,Silver P A.Genome-wide RNAi screen discovers functional coupling of alternative splicing and cell cycle control to apoptosis regulation.Cell Cycle,2010,9:4419–4421
15 Gu L,Guo R.Genome-wide detection and analysis of alternative splicing for nucleotide binding site-leucine-rich repeats sequences in rice.J genet genomics,2007,34:247–257
16 Leviatan N,Alkan N,Leshkowitz D,et al.Genome-wide survey of cold stress regulated alternative splicing in Arabidopsis thaliana with tiling microarray.PLo S One,2013,8:e66511
17 Staiger D,Brown J W.Alternative splicing at the intersection of biological timing,development,and stress responses.Plant Cell,2013,25 :3640–3656
18 Quesada V,Macknight R,Dean C,et al.Autoregulation of FCA pre-m RNA processing controls Arabidopsis flowering time.EMBO J,2003,22:3142–3152
19 Ding F,Cui P,Wang Z Y,et al.Genome-wide analysis of alternative splicing of pre-m RNA under salt stress in Arabidopsis.BMC Genomics,2014,15:431
20 Wang E T,Sandberg R,Luo S,et al.Alternative isoform regulation in human tissue transcriptomes.Nature,2008,456:470–476
21 Filichkin S A,Priest H D,Givan S A,et al.Genome-wide mapping of alternative splicing in Arabidopsis thaliana.Genome Res,2010,20:45 –58
22 Goldberg D H,Victor J D,Gardner E P,et al.Spike train analysis toolkit:enabling wider application of information-theoretic techniques to neurophysiology.Neuroinformatics,2009,7:165–178
23 Patel R K,Jain M.NGS QC Toolkit:a toolkit for quality control of next generation sequencing data.PLo S One,2012,7:e30619
24 Trapnell C,Pachter L,Salzberg S L.Top Hat:discovering splice junctions with RNA-Seq.Bioinformatics,2009,25:1105–1111
25 Quevillon E,Silventoinen V,Pillai S,et al.Inter Pro Scan:protein domains identifier.Nucleic Acids Res,2005,33:W116–W120
26 Trapnell C,Roberts A,Goff L,et al.Differential gene and transcript expression analysis of RNA-seq experiments with Top Hat and Cufflinks.Nat Protoc,2012,7:562–578
27 Rabbitts T H.Evidence for splicing of interrupted immunoglobulin variable and constant region sequences in nuclear RNA.Nature,1978,275 :291–296
28 Black D L.Mechanisms of alternative pre-messenger RNA splicing.Annu Rev Biochem,2003,72:291–336
29 Stamm S,Ben-Ari S,Rafalska I,et al.Function of alternative splicing.Gene,2005,344:1–20
30 Lareau L F,Green R E,Bhatnagar R S,et al.The evolving roles of alternative splicing.Curr Opin Struc Biol,2004,14:273–282
31 Xu Q,Modrek B,Lee C.Genome-wide detection of tissue-specific alternative splicing in the human transcriptome.Nucleic Acids Res,2002,30:3754–3766
32 Transcriptome and genome conservation of alternative splicing events in humans and mice.In:Sugnet C W,Kent W J,Haussler D,eds.Proc.9th Pacific Symposium on Biocomputing.Hawaii.2004,Singapore:World Scientific,2004.66–77
33 Chacko E,Ranganathan S.Genome-wide analysis of alternative splicing in cow:implications in bovine as a model for human diseases.BMC Genomics,2009,10:S11
34 Babenko V N,Aitnazarov R B,Goncharov F A,et al.Alternative splicing landscape of the Drosophila melanogaster genome.Russ J Genet,2010,46:1036–1038
35 Labadorf A,Link A,Rogers M F,et al.Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii.BMC Genomics,2010,11:114
36 Ramani A K,Calarco J A,Pan Q,et al.Genome-wide analysis of alternative splicing in Caenorhabditis elegans.Genome Res,2011,21:342 –348
37 Sablok G,Gupta P K,Baek J M,et al.Genome-wide survey of alternative splicing in the grass Brachypodium distachyon:a emerging model biosystem for plant functional genomics.Biotechnol Lett,2011,33:629–636
38 Bao H,Li E Y,Mansfield S D,et al.The developing xylem transcriptome and genome-wide analysis of alternative splicing in Populus trichocarpa(black cottonwood)populations.BMC Genomics,2013,14:359
39 Panahi B,Abbaszadeh B,Taghizadeghan M,et al.Genome-wide survey of alternative splicing in Sorghum Bicolor.Physiol Mol Biol Plants,2014,20:323–329
40 Wu H P,Su Y S,Chen H C,et al.Genome-wide analysis of light-regulated alternative splicing mediated by photoreceptors in Physcomitrella patens.Genome Biol,2014,15:R10
41 Shen Y,Zhou Z,Wang Z,et al.Global dissection of alternative splicing in paleopolyploid soybean.Plant Cell,2014,26:996–1008
42 Li P,Ponnala L,Gandotra N,et al.The developmental dynamics of the maize leaf transcriptome.Nat Genet,2010,42:1060–1067
43 Marquez Y,Brown J W,Simpson C,et al.Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis.Genome Res,2012,22:1184–1195
44 Pan Q,Shai O,Lee L J,et al.Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.Nat Genet,2008,40:1413–1415
45 Walters B,Lum G,Sablok G,et al.Genome-wide landscape of alternative splicing events in Brachypodium distachyon.DNA Res,2013,20 :163–171
46 Baek J M,Han P,Iandolino A,et al.Characterization and comparison of intron structure and alternative splicing between Medicago truncatula,Populus trichocarpa,Arabidopsis and rice.Plant Mol Biol,2008,67:499–510
47 Staiger C J.Signaling to the actin cytoskeleton in plants.Annu Rev Plant Physiol Plant Mol Biol,2000,51:257–288
48 Kadota A,Wada M.Photoorientation of chloroplasts in protonemal cells of the fern Adiantum as analyzed by use of a video-tracking system.Bot Mag,1992,105:265–279
49 Wada M,Kagawa T,Sato Y.Chloroplast movement.Annu Rev Plant Biol,2003,54:455–468
50 Stock A M,Robinson V L,Goudreau P N.Two-component signal transduction.Annu Rev Biochem,2000,69:183–215
51 吴家森,管剑峰.秦美猕猴桃果实生育及营养量变的若干特点.浙江林学院学报,2002,19:244–246
52 安华明,樊卫国,刘进平.生育期猕猴桃果实中营养元素积累规律研究.种子,2003,4:24–25
53 陈昆松.ABA和IAA对猕猴桃果实成熟进程的调控.园艺学报,1999,26:81–86
54 张玉,陈昆松,张上隆,等.猕猴桃果实采后成熟过程中糖代谢及其调节.植物生理与分子生物学学报,2004,30:317–324
55 涂正顺,李华,王华,等.猕猴桃果实采后香气成分的变化.园艺学报,2001,28:512–516
56 殷学仁,张波,李鲜,等.乙烯信号转导与果实成熟衰老的研究进展.园艺学报,2009,36:133–140