基于Illumina HiSeq 2500测序技术对高温胁迫下苦瓜叶片转录组特性分析
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摘要
高温胁迫时影响苦瓜作物产量及地域分布的重要限制因子,但由于其在非生物胁迫逆境方面的生物学信息研究进展较慢,这在一定程度上制约了苦瓜分子育种及品种改良工作。利用Illumina HiSeqTM2500高通量测序平台对高温胁迫处理前后的苦瓜叶片进行转录组测序,结果组装获得平均长度为700.75 bp共89 757条有效Unigene,N50长度达到1 239 bp;有48 662条Unigene获得注释信息,占全部Unigene的54.2%,与未处理的对照相比,共有2 254差异表达基因,其中672个上调,1 582下调表达。有27 116条Unigene能够注释到GO数据库,获得包含于细胞组分、分子功能和生物学过程三大类内共135 967个GO的功能注释,并且差异表达基因的GO功能注释分类情况大致与之相似。进一步筛选到可能与高温胁迫相关的基因,包括HSP,HSF等家族基因。本研究是首次对高温胁迫下的苦瓜叶片转录组进行分析,为进一步开展苦瓜耐高温分子生物学层面研究提供大量参考信息。
High temperature(HT) stress is an important factor to affect the yield and the quality of Momordica charantia L. However, the progress of biological information in abiotic stress is slow, which restricts molecular breeding and variety improvement of Momordica charantia to some extent. Transcriptome sequencing of Momordica charantia leaves before and after high temperature stress using Illumina HiSeqTM2500 high-throughput sequencing platform. The results indicated that 89 757 Unigene were obtained through sequence assembly with the average length was 700.75 bp and the length of N50 was 1 239 bp. 48 662 Unigene were annotated, accounting for 54.2%of the total Unigene. Compared with the untreated control, there were 2 254 differentially expressed genes, of which 672 were up-regulated and 1 582 down-regulated. There were 27 116 Unigene annotations to the GO database.A nd a total of 135 967 GO annotation functions were obtained, which were classified into three categories ofcellular components, molecular functions and biological processes. GO classification of differentia lly expre ssed genes was similarly. Genes that might be related to high temperature stress, including family genes such as HSP,HSF, were further screened. This study analyzed the transcriptome of Momordica charantia L. under HT providing a large amount of data source for the further study of molecular biology of Momordica charantia L. at the first time.
High temperature(HT) stress is an important factor to affect the yield and the quality of Momordica charantia L. However, the progress of biological information in abiotic stress is slow, which restricts molecular breeding and variety improvement of Momordica charantia to some extent. Transcriptome sequencing of Momordica charantia leaves before and after high temperature stress using Illumina HiSeqTM2500 high-throughput sequencing platform. The results indicated that 89 757 Unigene were obtained through sequence assembly with the average length was 700.75 bp and the length of N50 was 1 239 bp. 48 662 Unigene were annotated, accounting for 54.2%of the total Unigene. Compared with the untreated control, there were 2 254 differentially expressed genes, of which 672 were up-regulated and 1 582 down-regulated. There were 27 116 Unigene annotations to the GO database.A nd a total of 135 967 GO annotation functions were obtained, which were classified into three categories ofcellular components, molecular functions and biological processes. GO classification of differentia lly expre ssed genes was similarly. Genes that might be related to high temperature stress, including family genes such as HSP,HSF, were further screened. This study analyzed the transcriptome of Momordica charantia L. under HT providing a large amount of data source for the further study of molecular biology of Momordica charantia L. at the first time.
引文
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Chen X.F.,Huang R.K.,Hung Y.H.,Luo H.L.,and Chen Z.D.,2011,Advances in bitter gourd(Momordica charantia L.)breeding and related basic research,Nanfang Nongye Xuebao(Journal of Southern Agriculture),42(3):246-249(陈小凤,黄如葵,黄玉辉,罗海玲,陈振东,2011,苦瓜育种及相关基础研究进展,南方农业学报,42(3):246-249)
Florea L.,Song L.,and Salzberg S.L.,2013,Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues,F1000res,2:188
GullìM.,Rampino P.,Lupotto E.,Marmirol N.,and Perrotta C.,2005,The effect of heat stress and cadmium ions on the expression of a small hsp gene in barley and maize,J.Cereal Sci.,42(1):25-31
Guo P.G.,Li R.H.,Xia Y.S.,Zheng Y.S.,Miao S.Y.,Chen J.H.,and Zhang H.,2013,Effects of high temperature stress on several physiological traits in Momordica charantia,Guangzhou Daxue Xuebao(Journal of Guangzhou University(Natural Science Edition)),12(2):24-29(郭培国,李荣华,夏岩石,郑岩松,缪绅裕,陈健辉,张华,2013,高温胁迫对苦瓜生理特性影响的分析,广州大学学报(自然科学版),12(2):24-29)
Guo S.J.,Zhou H.Y.,Zhang X.S.,Li X.G.,and Meng Q.W.,2007,Overexpression of CaHSP26 in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance,J.Plant Physiol.,164(2):126-136
Jiang X.M.,Wu Y.F.,Xiao F.M.,Xiong Z.Y.,and Xu H.N.,Transcriptome analysis for leaves of five chemical types in Cinnamomum camphora,Yichuan(Hereditas),36(1):58-68(江香梅,伍艳芳,肖复明,熊振宇,徐海宁,2014,樟树5种化学类型叶片转录组分析,遗传,36(1):58-68)
Liu K.L.,and Chen W.G.,2015,Recent advances in plant heat-related genes,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),16(1):127-132(刘克禄,陈卫国,2015,植物耐热相关基因研究进展,植物遗传资源学报,16(1):127-132)
Liu X.Y.,2006,Isolation and functional analysis of LeFAD7 gene in tomato under temperature stress,Dissertation for Ph.D.,Shandong Agriculture University,Supervisor:Meng Q.W.,pp.13-27(刘训言,2006,番茄叶绿体ω-3脂肪酸去饱和酶基因(LeFAD7)的克隆及其在温度逆境下的功能分析,博士学位论文,山东农业大学,导师:孟庆伟,pp.13-27)
Liu Y.F.,Wan H.J.,Yang Y.J.,Wei Y.P.,Li Z.M.,Ye Q.J.,Wang R.Q.,Ruan M.Y.,Yao Z.P.,and Zhou G.Z.,2014,Genomewide identification and analysis of heat shock protein 90 in tomato,Yichuan(Hereditas),36(10):1043-1052(刘云飞,万红建,杨悦俭,韦艳萍,李志邈,叶青静,王荣青,阮美颖,姚祝平,周国治,2014,番茄热激蛋白90的全基因组鉴定及分析,遗传,36(10):1043-1052)
Luan F.S.,Lu B.Y.,Zhou H.W.,and Wang X.Z.,2016,Construction of genetic linkage map and QTL location for fruit-related traits in melon,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),47(8):9-20(栾非时,卢丙洋,周慧文,王学征,2016,甜瓜遗传连锁图谱构建及果实相关性状QTL定位,东北农业大学学报,47(8):9-20)
Murakami Y.,Tsuyama M.,Kobayashi Y.,Kodama H.,and Iba K.,2000,Trienoic fatty acids and plant tolerance of high temperature,Science,287(5452):476-479
Netasharir I.,Isaacson T.,Lurie S.,and Weiss D.,2005,Dual role for tomato heat shock protein 21:protecting photosystemⅡfrom oxidative stress and promoting color changes during fruit maturation,Plant Cell,17:1829-1838
Park S.M.,and Hong C.B.,2002,Class I small heat-shock protein gives thermotolerance in tobacco,J.Plant Physiol.,159(1):25-30
Pu M.,Lian X.P.,Luo S.L.,Zhang H.C.,Wang Y.K.,Bai X.J,,Zeng J.,Gao Q.G.,Ren X.S.,and Zhu L.Q.,2018,Transcriptome analysis of stigma after pollination in Brassica oleracea var capitata,Yuanyi Xuebao(Acta Horticulturae S-inica),45(1):71-84(蒲敏,廉小平,罗绍兰,张贺翠,王玉奎,白晓璟,曾静,高启国,任雪松,朱利泉,2018,结球甘蓝自花和异花授粉诱导的柱头转录组分析,园艺学报,45(1):71-84)
Rhoads D.M.,White S.J.,Zhou Y.,Muralidharan M.,and Elthon T.E.,2010,Altered gene expression in plants with constitutive expression of a mitochondrial small heat shock protein suggests the involvement of retrograde regulation in the heat stress response,Physiol.Plant.,123(4):435-444
Salunkhe D.K.,and Kadam S.S.,1998,Handbook of vegetable science and technology,production,composition,storage,and processing,PLoS One,5(12):e14323
Sanmiya K.,Suzuki K.,Egawa Y.,and Shono M.,2004,Mitochondrial small heat-shock protein enhances thermotolerance in tobacco plants,FEBS Lett.,557(1-3):265-268
Trapnell C.,Williams B.A.,Pertea G.,Mortazavi A.,Kwan G.,van Baren M.J.,Salzberg S.L.,Wold B.J.,and Pachter L.,2010,Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation,Nat.Biotechnol.,28(5):511-515
Xu X.M.,Wang R.,Wu T.Q.,He X.M.,and Lin Y.E.,2016,Screening of CsWRKYs involved in phytophthora blight resistance in cucumber(Cucumis sativus),Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),37(2):376-383(徐晓美,王瑞,吴廷全,何晓明,林毓娥,2016,对瓜类疫霉菌诱导响应的黄瓜WRKY转录因子筛选研究,热带作物学报,37(2):376-383)
Yang J.Y.,Sun Y.,Sun A.Q.,Yi S.Y.,Qin J.,Li M.H.,and Liu J.,2006,The involvement of chloroplast HSP100/ClpB in the acquired thermotolerance in tomato,Plant Mol.Biol.,62(3):385-395
Yi B.,2014,Transcriptional analysis of tomato under high temperature stress via solexa sequencing and cloning and functional analysis of LeCOR413-Tm1 gene,Thesis for M.S.,Shandong Agriculture University,Supervisor:Meng Q.W.,pp.5-10(尹波,2014,高温胁迫下番茄Solexa转录组测序及LeCOR413-TM1基因的克隆和功能分析,硕士学位论文,山东农业大学,导师:孟庆伟,pp.5-10)
Yokotani N.,Ichikawa T.,Kondou Y.,Matsui M.,Hirochika H.,Iwabuchi M.,and Oda K.,2008,Expression of rice heat stress transcription factor Os Hsf A2e enhances tolerance to environmental stresses in transgenic Arabidopsis,Planta,227(5):957-967
Zeng J.,Zhai Y.F.,Wu Z.M.,and Hu K.L.,2011,Effects of high temperature in summer on yield and its components in bitter gourd with 15 cross combinations and strains,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),32(11):2025-2028(曾晶,翟英芬,吴智明,胡开林,2011,夏季高温对15个苦瓜杂交组合(品系)产量及其构成性状的影响,热带作物学报,32(11):2025-2028)
Zhang D.S.,Zhao H.,Zhang F.L.,Yu Y.J.,Zhao X.Y.,Yu S.C.,Wang W.H.,Su T.B.,and Lu G.X.,2017,Identification and RNA-seq of new purple Chinese cabbage 15NG28 proge nies,Huabei Nongxuebao(Acta Agriculturae Boreali Sinica),32(6):14-24(张德双,赵泓,张凤兰,余阳俊,赵岫云,于拴仓,汪维红,苏同兵,卢桂香,2017,新型紫色大白菜15NG28后代的鉴别及其转录组分析,华北农学报,32(6):14-24)
Zhang S.P.,Qiu S.L.,Zheng Y.Y.,Zhang S.,Wu S.H.,He Y.S.,and Zheng K.B.,2017,The purple Abelmoschus esculentus transcriptome as a source for gene sequence information,Henongxue Bao(Journal of Nuclear Agricultural Sciences),31(4):643-653(张少平,邱珊莲,郑云云,张帅,吴松海,何炎森,郑开斌,2017,紫色黄秋葵转录组功能基因测序及分析,核农学报,31(4):643-653)
Anders S.,and Huber W.,2010,Differential expression analysis for sequence count data,Genome Biol.,11(10):R106
Chen X.F.,Huang R.K.,Hung Y.H.,Luo H.L.,and Chen Z.D.,2011,Advances in bitter gourd(Momordica charantia L.)breeding and related basic research,Nanfang Nongye Xuebao(Journal of Southern Agriculture),42(3):246-249(陈小凤,黄如葵,黄玉辉,罗海玲,陈振东,2011,苦瓜育种及相关基础研究进展,南方农业学报,42(3):246-249)
Florea L.,Song L.,and Salzberg S.L.,2013,Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues,F1000res,2:188
GullìM.,Rampino P.,Lupotto E.,Marmirol N.,and Perrotta C.,2005,The effect of heat stress and cadmium ions on the expression of a small hsp gene in barley and maize,J.Cereal Sci.,42(1):25-31
Guo P.G.,Li R.H.,Xia Y.S.,Zheng Y.S.,Miao S.Y.,Chen J.H.,and Zhang H.,2013,Effects of high temperature stress on several physiological traits in Momordica charantia,Guangzhou Daxue Xuebao(Journal of Guangzhou University(Natural Science Edition)),12(2):24-29(郭培国,李荣华,夏岩石,郑岩松,缪绅裕,陈健辉,张华,2013,高温胁迫对苦瓜生理特性影响的分析,广州大学学报(自然科学版),12(2):24-29)
Guo S.J.,Zhou H.Y.,Zhang X.S.,Li X.G.,and Meng Q.W.,2007,Overexpression of CaHSP26 in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance,J.Plant Physiol.,164(2):126-136
Jiang X.M.,Wu Y.F.,Xiao F.M.,Xiong Z.Y.,and Xu H.N.,Transcriptome analysis for leaves of five chemical types in Cinnamomum camphora,Yichuan(Hereditas),36(1):58-68(江香梅,伍艳芳,肖复明,熊振宇,徐海宁,2014,樟树5种化学类型叶片转录组分析,遗传,36(1):58-68)
Liu K.L.,and Chen W.G.,2015,Recent advances in plant heat-related genes,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),16(1):127-132(刘克禄,陈卫国,2015,植物耐热相关基因研究进展,植物遗传资源学报,16(1):127-132)
Liu X.Y.,2006,Isolation and functional analysis of LeFAD7 gene in tomato under temperature stress,Dissertation for Ph.D.,Shandong Agriculture University,Supervisor:Meng Q.W.,pp.13-27(刘训言,2006,番茄叶绿体ω-3脂肪酸去饱和酶基因(LeFAD7)的克隆及其在温度逆境下的功能分析,博士学位论文,山东农业大学,导师:孟庆伟,pp.13-27)
Liu Y.F.,Wan H.J.,Yang Y.J.,Wei Y.P.,Li Z.M.,Ye Q.J.,Wang R.Q.,Ruan M.Y.,Yao Z.P.,and Zhou G.Z.,2014,Genomewide identification and analysis of heat shock protein 90 in tomato,Yichuan(Hereditas),36(10):1043-1052(刘云飞,万红建,杨悦俭,韦艳萍,李志邈,叶青静,王荣青,阮美颖,姚祝平,周国治,2014,番茄热激蛋白90的全基因组鉴定及分析,遗传,36(10):1043-1052)
Luan F.S.,Lu B.Y.,Zhou H.W.,and Wang X.Z.,2016,Construction of genetic linkage map and QTL location for fruit-related traits in melon,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),47(8):9-20(栾非时,卢丙洋,周慧文,王学征,2016,甜瓜遗传连锁图谱构建及果实相关性状QTL定位,东北农业大学学报,47(8):9-20)
Murakami Y.,Tsuyama M.,Kobayashi Y.,Kodama H.,and Iba K.,2000,Trienoic fatty acids and plant tolerance of high temperature,Science,287(5452):476-479
Netasharir I.,Isaacson T.,Lurie S.,and Weiss D.,2005,Dual role for tomato heat shock protein 21:protecting photosystemⅡfrom oxidative stress and promoting color changes during fruit maturation,Plant Cell,17:1829-1838
Park S.M.,and Hong C.B.,2002,Class I small heat-shock protein gives thermotolerance in tobacco,J.Plant Physiol.,159(1):25-30
Pu M.,Lian X.P.,Luo S.L.,Zhang H.C.,Wang Y.K.,Bai X.J,,Zeng J.,Gao Q.G.,Ren X.S.,and Zhu L.Q.,2018,Transcriptome analysis of stigma after pollination in Brassica oleracea var capitata,Yuanyi Xuebao(Acta Horticulturae S-inica),45(1):71-84(蒲敏,廉小平,罗绍兰,张贺翠,王玉奎,白晓璟,曾静,高启国,任雪松,朱利泉,2018,结球甘蓝自花和异花授粉诱导的柱头转录组分析,园艺学报,45(1):71-84)
Rhoads D.M.,White S.J.,Zhou Y.,Muralidharan M.,and Elthon T.E.,2010,Altered gene expression in plants with constitutive expression of a mitochondrial small heat shock protein suggests the involvement of retrograde regulation in the heat stress response,Physiol.Plant.,123(4):435-444
Salunkhe D.K.,and Kadam S.S.,1998,Handbook of vegetable science and technology,production,composition,storage,and processing,PLoS One,5(12):e14323
Sanmiya K.,Suzuki K.,Egawa Y.,and Shono M.,2004,Mitochondrial small heat-shock protein enhances thermotolerance in tobacco plants,FEBS Lett.,557(1-3):265-268
Trapnell C.,Williams B.A.,Pertea G.,Mortazavi A.,Kwan G.,van Baren M.J.,Salzberg S.L.,Wold B.J.,and Pachter L.,2010,Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation,Nat.Biotechnol.,28(5):511-515
Xu X.M.,Wang R.,Wu T.Q.,He X.M.,and Lin Y.E.,2016,Screening of CsWRKYs involved in phytophthora blight resistance in cucumber(Cucumis sativus),Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),37(2):376-383(徐晓美,王瑞,吴廷全,何晓明,林毓娥,2016,对瓜类疫霉菌诱导响应的黄瓜WRKY转录因子筛选研究,热带作物学报,37(2):376-383)
Yang J.Y.,Sun Y.,Sun A.Q.,Yi S.Y.,Qin J.,Li M.H.,and Liu J.,2006,The involvement of chloroplast HSP100/ClpB in the acquired thermotolerance in tomato,Plant Mol.Biol.,62(3):385-395
Yi B.,2014,Transcriptional analysis of tomato under high temperature stress via solexa sequencing and cloning and functional analysis of LeCOR413-Tm1 gene,Thesis for M.S.,Shandong Agriculture University,Supervisor:Meng Q.W.,pp.5-10(尹波,2014,高温胁迫下番茄Solexa转录组测序及LeCOR413-TM1基因的克隆和功能分析,硕士学位论文,山东农业大学,导师:孟庆伟,pp.5-10)
Yokotani N.,Ichikawa T.,Kondou Y.,Matsui M.,Hirochika H.,Iwabuchi M.,and Oda K.,2008,Expression of rice heat stress transcription factor Os Hsf A2e enhances tolerance to environmental stresses in transgenic Arabidopsis,Planta,227(5):957-967
Zeng J.,Zhai Y.F.,Wu Z.M.,and Hu K.L.,2011,Effects of high temperature in summer on yield and its components in bitter gourd with 15 cross combinations and strains,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),32(11):2025-2028(曾晶,翟英芬,吴智明,胡开林,2011,夏季高温对15个苦瓜杂交组合(品系)产量及其构成性状的影响,热带作物学报,32(11):2025-2028)
Zhang D.S.,Zhao H.,Zhang F.L.,Yu Y.J.,Zhao X.Y.,Yu S.C.,Wang W.H.,Su T.B.,and Lu G.X.,2017,Identification and RNA-seq of new purple Chinese cabbage 15NG28 proge nies,Huabei Nongxuebao(Acta Agriculturae Boreali Sinica),32(6):14-24(张德双,赵泓,张凤兰,余阳俊,赵岫云,于拴仓,汪维红,苏同兵,卢桂香,2017,新型紫色大白菜15NG28后代的鉴别及其转录组分析,华北农学报,32(6):14-24)
Zhang S.P.,Qiu S.L.,Zheng Y.Y.,Zhang S.,Wu S.H.,He Y.S.,and Zheng K.B.,2017,The purple Abelmoschus esculentus transcriptome as a source for gene sequence information,Henongxue Bao(Journal of Nuclear Agricultural Sciences),31(4):643-653(张少平,邱珊莲,郑云云,张帅,吴松海,何炎森,郑开斌,2017,紫色黄秋葵转录组功能基因测序及分析,核农学报,31(4):643-653)