一个毛竹LTR转座子PHRE9的全长鉴定与转录活性分析
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摘要
反转录转座子作为毛竹(Phyllostachys edulis)基因组中重要组成部分,可以通过自身转座参与毛竹生长发育的调控,获得具有活性的毛竹转座子,对竹子突变育种有着重要意义。本研究鉴定了1条结构完整的毛竹LTR反转录转座子PHRE9 (Phyllostachys edulis retrotransposons 9),系统地分析了PHRE9结构特征和进化模式以及在逆境下的表达模式。通过对PHRE9转座子全长PCR扩增以及生物信息学分析,表明PHRE9全长为6 370 bp,属于Ty1-copia大类中的Tork分支。利用qRT-PCR技术检测了在DNA甲基化抑制剂、辐照、高盐、高温、低温等不同处理条件下,PHRE9的3个结构域的基因转录活性水平。结果表明在DNA甲基化抑制剂处理、辐照、高盐、高温(42℃)、低温(4℃)胁迫处理下PHRE9表达水平均有上调的现象,表现出转录激活特性。本研究为毛竹逆境适应机制研究以及竹子突变育种提供了基础资料。
Retrotransposons, as important parts of Phyllostachys edulis genome, participate in the regulation of the growth and development of Ph. edulis through their own transposonsition, and it is of great significance to obtain active transposons for the molecular breeding of Ph. edulis. In this study, a complete LTR retrotransposons from the Ph. edulis genome that named PHRE9 was characterized. The PCR amplification and bioinformatics analysis of the full length retrotransposon of PHRE9 show that the structural characteristics and evolution patterns as well as the expression patterns under adversity was systematically analyzed. The length of PHRE9 is 6 370 bp, belonging to the Tork branch in Ty1-copia super-family. The transcriptional activity levels of genes in three domains of PHRE9 was measured by qRT-PCR under different treatment conditions, such as DNA methylation inhibitors, irradiation, high salt, high temperature and low temperature.The results showed that PHRE9 expression level was raised under the stress of DNA methylation inhibitors treatment, irradiation, high salt, high temperature(42 ℃), low temperature(4 ℃). The results showed that PHRE9 was a transcription-active transposon. This study could provide basis data for the study of adversity adaptation mechanism and molecular breeding of Ph. edulis.
Retrotransposons, as important parts of Phyllostachys edulis genome, participate in the regulation of the growth and development of Ph. edulis through their own transposonsition, and it is of great significance to obtain active transposons for the molecular breeding of Ph. edulis. In this study, a complete LTR retrotransposons from the Ph. edulis genome that named PHRE9 was characterized. The PCR amplification and bioinformatics analysis of the full length retrotransposon of PHRE9 show that the structural characteristics and evolution patterns as well as the expression patterns under adversity was systematically analyzed. The length of PHRE9 is 6 370 bp, belonging to the Tork branch in Ty1-copia super-family. The transcriptional activity levels of genes in three domains of PHRE9 was measured by qRT-PCR under different treatment conditions, such as DNA methylation inhibitors, irradiation, high salt, high temperature and low temperature.The results showed that PHRE9 expression level was raised under the stress of DNA methylation inhibitors treatment, irradiation, high salt, high temperature(42 ℃), low temperature(4 ℃). The results showed that PHRE9 was a transcription-active transposon. This study could provide basis data for the study of adversity adaptation mechanism and molecular breeding of Ph. edulis.
引文
郭红媛,贾举庆,张莉,等.2014.燕麦EST数据库中Ty1-co?pia和Ty3-gypsy型反转录转座子的频数分析[J].中国农业大学学报,19(04):23-30.(Guo H Y,Jia J Q,Zhang L,et al.2014.Frequencies of Ty1-copia and Ty3-gypsy retroelement within the Avena EST database[J].Journal of China Agricultural University,19(04):23-30.)
李聪.2014.冷胁迫下铁皮石斛转录组及Ty1-copia类反转录转座子表达分析[D].硕士学位论文,浙江农林大学,导师:斯金平,pp.61-64.(Li C.2014.Ttanscriptom of Dendrobium officinale under cold stress and isolation and expression analysis of Ty1-copia group retrotransposons[D].Thesis for M.S.,Zhejiang A&F University,Supervisor:Si J P,pp.61-64.)
梁琳琳,周明兵.2016.植物活性长末端重复序列反转录转座子研究进展[J].生物工程学报,32(04):409-429.(Liang L L,Zhou M B.2016.Plant active LTR retrotransposons:A review[J].Chinese Journal of Biotechnology,32(04):409-429.)
刘静.2017.毛竹三个全长LTR转座子的分布、进化模式分析及PHRE5的克隆与功能鉴定[D].硕士学位论文,浙江农林大学,导师:周明兵,pp.12-68.(Liu J.2017.The analysis of distribution and evolution pattern of three full-longth LTR retrotransposons and characteriation and functional analysis of PHRE5 from Phyllostachys edulis[D].Thesis for M.S.,Zhejiang A&F University,Supervisor:Zhou M B,pp.12-68.)
刘炜.2006.干旱胁迫诱导的水稻基因组胞嘧啶甲基化变化[D].硕士学位论文,东北师范大学,导师:刘宝,pp.16-19.(Li W.2006.Drought-induced alterations in cytosine methylation in the rice genome[D].Thesis for M.S.,Northeast Normal University,Supervisor:Liu B,pp.16-19.)
马赑.2014.桑树全基因组转座子的鉴定及特征分析[D].博士学位论文,西南大学,导师:何宁佳,pp.96-102.(Ma B.2014.Genome-wide identification and characterization of transposable elements in mulberry(Morus notabi?lis)[D].Thesis for Ph.D.,Southwest University,Supervisor:He N J,pp.96-102.)
齐飞艳,胡陶,彭镇华,等.2013.毛竹实时荧光定量PCR内参基因的筛选及成花基因PheTFL1表达分析[J].西北植物学报,33(01):48-52.(Qi F Y,Hu T,Peng Z H,et al.2013.Screening of reference genes used in qRT-PCRand expression analysis of PheTFL1 gene in moso bamboo[J].Acta Botanica Boreali-Occidentalia Sinica,33(01):48-52.)
王丽丽,赵韩生,孙化雨,等.2015.胁迫条件下毛竹miR164b及其靶基因PeNAC1表达研究[J].林业科学研究,28(5):605-611.(Wang L L,Zhao H S,Sun H Y,et al.2015.Expression analysis of miR164b and its target gene PeNAC1 in Phyllostachys edulis under stress[J].Forest Research,28(5):605-611.)
王林红.2014.大豆干旱应激相关基因的克隆及分析[D].硕士学位论文,河北科技师范学院,导师:李桂兰,pp.16-19.(Wang L H.2014.Cloning and analysis of drought stress genes soybean[D].Thesis for M.S.,Hebei Normal University Of Science&Technology,Supervisor:Li GL,pp.16-19.)
徐小万,雷建军,张长远,等.2014.高温多湿胁迫下辣椒DNA甲基化分析[J].核农学报,28(07):1175-1180.(Xu X W,Lei J J,Zhang C Y,et al.2014.Methylationsensitive amplified polymorphism analysis of DNAmethylation in hot pepper under high temperature and air humidity stress[J].Journal of Nuclear Agricultural Sciences,28(07):1175-1180.)
杨震,郭会君,赵林姝,等.2015.60Co-γ射线诱导的小麦基因组DNA的甲基化变异[J].核农学报,29(1):0001-0009(YANG Z,GUO H J,ZHAO L S,et al.2015.The variation of DNA cytosine methylation in wheat genome induced by60Co-γrays[J].Journal of Nuclear Agricultural Sciences,29(1):0001-0009)
张赞一,周明兵,汤定钦.2018.毛竹LTR反转录转座子PHRE6的克隆与转录活性分析[J].中国细胞生物学学报,40(9):1466-1478.(Zhang Z Y,Zhou M B,Tang D Q.2018.Phyllostachys edulis LTR transposon-cloning and transcriptional activity identification of PHRE6[J].Chinese Journal of Cell Biology,40(9):1466-1478.)
赵慧茹,押辉远,谷运红,等.2009.60Co-γ辐照对小麦反转录转座子WIS2-1A表达活性的影响[J].河南农业科学,38(03):20-22.(Zhao H R,Ya H Y,Gu Y H,et al.2009.Effect of60Co-γirradiation on the expression activity of retrotransposon WIS2-1A[J].Journal of Henan Agricultural Sciences,38(03):20-22.)
Bender J.1998.Cytosine methylation of repeated sequences in eukaryotes:The role of DNA pairing[J].Trends in Biochemical Sciences,23(7):252-256.
Bennetzen J L.2000.Transposable element contributions to plant gene and genome evolution[J].Plant Molecular Biology,42(1):251-269.
Bennetzen J L.2005.Transposable elements,gene creation and genome rearrangement in flowering plants[J].Current Opinion in Genetics&Development,15(6):621-721.
Bowen N J,McDonald J F.2001.Drosophila euchromatic LTR retrotransposons are much younger than the host species in which they reside[J].Genome Research,11(9):1527-1540.
Bucher E,Reinders J,Mirouze M.2012.Epigenetic control of transposon transcription and mobility in Arabidopsis[J].Current Opinion in Plant Biology,15(5):503-510.
Choi C S,Sano H.2007.Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants[J].Molecular Genetics and Genomics,277(5):589-600.
Domingues D S,Cruz G M Q,Metcalfe C J,et al.2012.Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns[J].BMCGenomics,13(1):137-149.
Ellinghaus D,Kurtz S,Willhoeft U.2008.LTRharvest,an efficient and flexible software for de novo detection of LTRretrotransposons[J].BMC Bioinformatic,9(1):18-31.
Flavell A J,Pearce S R,Kumar A.1994.Plant transposable elements and the genome[J].Current Opinion in Genetics&Development,4(6):838-844.
Finnegan D J.2012.Retrotransposons[J].Current Biology,22(11):432-437.
Goll M G,Bestor T H.2005.Eukaryotic cytosine methyltransferases[J].Annual Review Biochemistry,74(1):481-514.
Kumar A,Bennetzen J L.1999.Plant retrotransposons[J].Annual Review of Genetics,33(1):479-532.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,25(4):402-802.
Lisch D.2013.How important are transposons for plant evolution[J].Nature Reviews Genetics,14(1):49-61.
Li W,Godzik A.2006.Cd-hit:A fast program for clustering and comparing large sets of protein or nucleotide sequences[J].Bioinformatics,22(13):1658-1659.
Ma J,Jackson S A.2006.Retrotransposon accumulation and satellite amplification mediated by segmental duplication facilitate centromere expansion in rice[J].Genome Research,16(2):251-259.
Makarevitch I,Waters A J,West P T,et al.2015.Transposable elements contribute to activation of maize genes in response to abiotic stress[J].Plos Genetics,11(1):e1004915.
Peng Z H,Lu Y,Li L B,et al.2013.The draft genome of the fast-growing non-timber forest species moso bamboo(Phyllostachys heterocycla)[J].Nature Genetics,45(4):456-461.
Rico-Cabanas L,Martínez-Izquierdo J A.2007.CIRE1,a novel transcriptionally active Ty1-copia retrotransposon from Citrus sinensis[J].Molecular Genetics&Genomics,277(4):365-377.
Sormacheva I D 2011.LTR retrotransposons in plants[J].Russian Journal of Genetics Applied Research,1(6):540-564.
Steward N,Ito M,Yamaguchi Y,et al.2002.Periodic DNAmethylation in maize nucleosomes and demethylation by environmental stress[J].Journalof Biological Chemistry,277(40):37741-37746.
Steward N,Kusano T,Sano H.2000.Expression of ZmMET1,a gene encoding a DNA methyltransferase from maize,is associated not only with DNA replication in actively proliferating cells,but also with altered DNA methylation status in cold-stressed quiescent cells[J].Nucleic Acids Research,28(17):3250-3259.
Steinbiss S,Willhoeft U,Gremme G,et al.2009.Fine-grained annotation and classification of de novo predicted LTRretrotransposons[J].Nucleic Acids Research,37(21):7002-7013.
Woodrow P,Pontecorvo G,Fantaccione S,et al.2010.Polymorphism of a new Ty1-copia retrotransposon in durum wheat under salt and light stresses[J].Theoretical and Applied Genetics,121(2):311-322.
Xiong Y,Eickbush T H.1990.Origin and evolution of retroelements based upon their reverse transcriptase sequences[J].The Embo Journal,9(10):3353-3362.
李聪.2014.冷胁迫下铁皮石斛转录组及Ty1-copia类反转录转座子表达分析[D].硕士学位论文,浙江农林大学,导师:斯金平,pp.61-64.(Li C.2014.Ttanscriptom of Dendrobium officinale under cold stress and isolation and expression analysis of Ty1-copia group retrotransposons[D].Thesis for M.S.,Zhejiang A&F University,Supervisor:Si J P,pp.61-64.)
梁琳琳,周明兵.2016.植物活性长末端重复序列反转录转座子研究进展[J].生物工程学报,32(04):409-429.(Liang L L,Zhou M B.2016.Plant active LTR retrotransposons:A review[J].Chinese Journal of Biotechnology,32(04):409-429.)
刘静.2017.毛竹三个全长LTR转座子的分布、进化模式分析及PHRE5的克隆与功能鉴定[D].硕士学位论文,浙江农林大学,导师:周明兵,pp.12-68.(Liu J.2017.The analysis of distribution and evolution pattern of three full-longth LTR retrotransposons and characteriation and functional analysis of PHRE5 from Phyllostachys edulis[D].Thesis for M.S.,Zhejiang A&F University,Supervisor:Zhou M B,pp.12-68.)
刘炜.2006.干旱胁迫诱导的水稻基因组胞嘧啶甲基化变化[D].硕士学位论文,东北师范大学,导师:刘宝,pp.16-19.(Li W.2006.Drought-induced alterations in cytosine methylation in the rice genome[D].Thesis for M.S.,Northeast Normal University,Supervisor:Liu B,pp.16-19.)
马赑.2014.桑树全基因组转座子的鉴定及特征分析[D].博士学位论文,西南大学,导师:何宁佳,pp.96-102.(Ma B.2014.Genome-wide identification and characterization of transposable elements in mulberry(Morus notabi?lis)[D].Thesis for Ph.D.,Southwest University,Supervisor:He N J,pp.96-102.)
齐飞艳,胡陶,彭镇华,等.2013.毛竹实时荧光定量PCR内参基因的筛选及成花基因PheTFL1表达分析[J].西北植物学报,33(01):48-52.(Qi F Y,Hu T,Peng Z H,et al.2013.Screening of reference genes used in qRT-PCRand expression analysis of PheTFL1 gene in moso bamboo[J].Acta Botanica Boreali-Occidentalia Sinica,33(01):48-52.)
王丽丽,赵韩生,孙化雨,等.2015.胁迫条件下毛竹miR164b及其靶基因PeNAC1表达研究[J].林业科学研究,28(5):605-611.(Wang L L,Zhao H S,Sun H Y,et al.2015.Expression analysis of miR164b and its target gene PeNAC1 in Phyllostachys edulis under stress[J].Forest Research,28(5):605-611.)
王林红.2014.大豆干旱应激相关基因的克隆及分析[D].硕士学位论文,河北科技师范学院,导师:李桂兰,pp.16-19.(Wang L H.2014.Cloning and analysis of drought stress genes soybean[D].Thesis for M.S.,Hebei Normal University Of Science&Technology,Supervisor:Li GL,pp.16-19.)
徐小万,雷建军,张长远,等.2014.高温多湿胁迫下辣椒DNA甲基化分析[J].核农学报,28(07):1175-1180.(Xu X W,Lei J J,Zhang C Y,et al.2014.Methylationsensitive amplified polymorphism analysis of DNAmethylation in hot pepper under high temperature and air humidity stress[J].Journal of Nuclear Agricultural Sciences,28(07):1175-1180.)
杨震,郭会君,赵林姝,等.2015.60Co-γ射线诱导的小麦基因组DNA的甲基化变异[J].核农学报,29(1):0001-0009(YANG Z,GUO H J,ZHAO L S,et al.2015.The variation of DNA cytosine methylation in wheat genome induced by60Co-γrays[J].Journal of Nuclear Agricultural Sciences,29(1):0001-0009)
张赞一,周明兵,汤定钦.2018.毛竹LTR反转录转座子PHRE6的克隆与转录活性分析[J].中国细胞生物学学报,40(9):1466-1478.(Zhang Z Y,Zhou M B,Tang D Q.2018.Phyllostachys edulis LTR transposon-cloning and transcriptional activity identification of PHRE6[J].Chinese Journal of Cell Biology,40(9):1466-1478.)
赵慧茹,押辉远,谷运红,等.2009.60Co-γ辐照对小麦反转录转座子WIS2-1A表达活性的影响[J].河南农业科学,38(03):20-22.(Zhao H R,Ya H Y,Gu Y H,et al.2009.Effect of60Co-γirradiation on the expression activity of retrotransposon WIS2-1A[J].Journal of Henan Agricultural Sciences,38(03):20-22.)
Bender J.1998.Cytosine methylation of repeated sequences in eukaryotes:The role of DNA pairing[J].Trends in Biochemical Sciences,23(7):252-256.
Bennetzen J L.2000.Transposable element contributions to plant gene and genome evolution[J].Plant Molecular Biology,42(1):251-269.
Bennetzen J L.2005.Transposable elements,gene creation and genome rearrangement in flowering plants[J].Current Opinion in Genetics&Development,15(6):621-721.
Bowen N J,McDonald J F.2001.Drosophila euchromatic LTR retrotransposons are much younger than the host species in which they reside[J].Genome Research,11(9):1527-1540.
Bucher E,Reinders J,Mirouze M.2012.Epigenetic control of transposon transcription and mobility in Arabidopsis[J].Current Opinion in Plant Biology,15(5):503-510.
Choi C S,Sano H.2007.Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants[J].Molecular Genetics and Genomics,277(5):589-600.
Domingues D S,Cruz G M Q,Metcalfe C J,et al.2012.Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns[J].BMCGenomics,13(1):137-149.
Ellinghaus D,Kurtz S,Willhoeft U.2008.LTRharvest,an efficient and flexible software for de novo detection of LTRretrotransposons[J].BMC Bioinformatic,9(1):18-31.
Flavell A J,Pearce S R,Kumar A.1994.Plant transposable elements and the genome[J].Current Opinion in Genetics&Development,4(6):838-844.
Finnegan D J.2012.Retrotransposons[J].Current Biology,22(11):432-437.
Goll M G,Bestor T H.2005.Eukaryotic cytosine methyltransferases[J].Annual Review Biochemistry,74(1):481-514.
Kumar A,Bennetzen J L.1999.Plant retrotransposons[J].Annual Review of Genetics,33(1):479-532.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtmethod[J].Methods,25(4):402-802.
Lisch D.2013.How important are transposons for plant evolution[J].Nature Reviews Genetics,14(1):49-61.
Li W,Godzik A.2006.Cd-hit:A fast program for clustering and comparing large sets of protein or nucleotide sequences[J].Bioinformatics,22(13):1658-1659.
Ma J,Jackson S A.2006.Retrotransposon accumulation and satellite amplification mediated by segmental duplication facilitate centromere expansion in rice[J].Genome Research,16(2):251-259.
Makarevitch I,Waters A J,West P T,et al.2015.Transposable elements contribute to activation of maize genes in response to abiotic stress[J].Plos Genetics,11(1):e1004915.
Peng Z H,Lu Y,Li L B,et al.2013.The draft genome of the fast-growing non-timber forest species moso bamboo(Phyllostachys heterocycla)[J].Nature Genetics,45(4):456-461.
Rico-Cabanas L,Martínez-Izquierdo J A.2007.CIRE1,a novel transcriptionally active Ty1-copia retrotransposon from Citrus sinensis[J].Molecular Genetics&Genomics,277(4):365-377.
Sormacheva I D 2011.LTR retrotransposons in plants[J].Russian Journal of Genetics Applied Research,1(6):540-564.
Steward N,Ito M,Yamaguchi Y,et al.2002.Periodic DNAmethylation in maize nucleosomes and demethylation by environmental stress[J].Journalof Biological Chemistry,277(40):37741-37746.
Steward N,Kusano T,Sano H.2000.Expression of ZmMET1,a gene encoding a DNA methyltransferase from maize,is associated not only with DNA replication in actively proliferating cells,but also with altered DNA methylation status in cold-stressed quiescent cells[J].Nucleic Acids Research,28(17):3250-3259.
Steinbiss S,Willhoeft U,Gremme G,et al.2009.Fine-grained annotation and classification of de novo predicted LTRretrotransposons[J].Nucleic Acids Research,37(21):7002-7013.
Woodrow P,Pontecorvo G,Fantaccione S,et al.2010.Polymorphism of a new Ty1-copia retrotransposon in durum wheat under salt and light stresses[J].Theoretical and Applied Genetics,121(2):311-322.
Xiong Y,Eickbush T H.1990.Origin and evolution of retroelements based upon their reverse transcriptase sequences[J].The Embo Journal,9(10):3353-3362.