蔗茅野生种响应低温胁迫的数字基因表达谱
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摘要
以蔗茅野生种(昆明蔗茅99-1)为材料,利用数字基因表达谱(DGE)技术,对4℃低温胁迫处理后的蔗茅野生种构建的c DNA文库进行差异基因表达谱分析。结果表明,低温处理24 h(ER-LT1)有2 391个基因,低温处理72 h(ER-LT2)有4 892个基因因低温处理而发生表达变化。GO功能显著性富集分析表明,差异基因主要涉及对非生物刺激反应、氧化还原过程、能量代谢以及催化活性。KEGG富集分析表明,两个不同处理时间下共同富集的通路主要为植物激素信号转导途径、植物-病原体互作通路、光合作用通路、淀粉和蔗糖代谢途径和苯丙烷生物合成途径等。利用实时荧光定量PCR(q RT-PCR)验证2个在低温胁迫条件下的差异表达基因,其结果与DGE分析结果基本一致,证实了DGE测序结果的可靠性。
Using the digital gene expression(DGE) technique, the differentially expressed genes were analyzed in the c DNA library constructed from the wild species of Erianthus fulvus named Ness 99-1, which were treated under 4℃. The results showed that 2 391 genes were induced at low temperature for 24 h(ER-LT1), and 4 892 genes were induced at low temperature for 72 h(ER-LT2). GO significant enrichment analysis indicated that the differential gene mainly related to response to abiotic stimulus, oxidation-reduction process, cellular carbohydrate metabolic process and catalytic activity. KEGG enrichment analysis suggested that the pathways of co-enrichment under two treatments mainly were plant hormone signal transduction pathway, plant-pathogen interaction pathway,photosynthetic pathway, starch and sucrose metabolic pathway and phenylalanine metabolism. Two differentially expressed genes under low temperature stress were validated by real-time quantitative PCR(q RT-PCR), the results of which were consistent with those of DGE and confirmed the reliability of DGE test results.
Using the digital gene expression(DGE) technique, the differentially expressed genes were analyzed in the c DNA library constructed from the wild species of Erianthus fulvus named Ness 99-1, which were treated under 4℃. The results showed that 2 391 genes were induced at low temperature for 24 h(ER-LT1), and 4 892 genes were induced at low temperature for 72 h(ER-LT2). GO significant enrichment analysis indicated that the differential gene mainly related to response to abiotic stimulus, oxidation-reduction process, cellular carbohydrate metabolic process and catalytic activity. KEGG enrichment analysis suggested that the pathways of co-enrichment under two treatments mainly were plant hormone signal transduction pathway, plant-pathogen interaction pathway,photosynthetic pathway, starch and sucrose metabolic pathway and phenylalanine metabolism. Two differentially expressed genes under low temperature stress were validated by real-time quantitative PCR(q RT-PCR), the results of which were consistent with those of DGE and confirmed the reliability of DGE test results.
引文
Audic S.,and Caverie J.M.,1997,The significance of digital gene expression profile,Genome Research,7(10):986-995
Bhardwaj A.R.,Joshi G.,Kukreja B.,Malik V.,Arora P.,Pandey R.,Shukla R.N.,Bankar K.G.,Agarwal S.K.,Goel S.,Jagannath A.,Kumar A.,and Agarwal M.,2015,Global insights into high temperature and drought stress regulated gen es by RNA-Seq in economically important oilseed crop Brassica juncea,BMC Plant Biol.,15(1):9
Fang Z.C.,Liu S.C.,Gao X.X.,Dao J.M.,and Fan X.,2014,Review on sugarcane stress physiology,Zhongguo Tangliao(Sugar Crops of China),39(4):79-82(方志存,刘少春,高欣欣,刀静梅,樊仙,2014,甘蔗逆境生理研究进展,中国糖料,39(4):79-82)
Hu H.L.,Hou X.M.,Qu B.,Gao X.J.,and Li Q.Z.,2012,Application of high throughput gene expression profiling,Zhongguo Ruping Gongye(China Dairy Industry),40(12):40-43(胡红柳,侯晓明,曲波,高学军,李庆章,2012,高通量基因表达谱的应用,中国乳品工业,40(12):40-43)
Huang X.,Chen M.H.,Yang L.T.,Zhang B.Q.,and Li Y.R.,2013,Effects of exogenous abscisic acid on cell membrane and endogenous hormones contents in leaves of sugarcane seedling under cold stress,Huazhong Nongye Daxue Xuebao(Journal of Huazhong Agricultural University),32(4):6-11(黄杏,陈明辉,杨丽涛,张保青,李杨瑞,2013,低温胁迫下外源ABA对甘蔗幼苗抗寒性及内源激素的影响,华中农业大学学报,32(4):6-11)
Li F.S.,Lin W.F.,and He S.C.,2004,Suggestions on developing and utilization of Erianthus fulvus wild species,Ziyuan Kaifa Yu Shichang(Resource Development&Market),20(4):266-270(李富生,林位夫,何顺长,2004,开发利用蔗茅野生种质资源的思考,资源开发与市场,20(4):266-270)
Li W.F.,Cai Q.,Huang Y.K.,Fan Y.H.,and Ma L.,2005,Identification of sugarcane wild germplasm resources resistant to Puccinia erianthi,Zhiwu Baohu(Plant Protection),31(2):51-53(李文凤,蔡青,黄应昆,范源洪,马丽,2005,甘蔗野生资源对蔗茅柄锈菌的抗性鉴定,植物保护,31(2):51-53)
Lin X.Q.,Liu X.,Liu X.L.,Mao J.,Zi Y.Q.,Liu H.B.,Li Z.L.,Li X.J.,Xu C.H.,Li C.J.,and Cai Q.,2016,Analysis of intergeneric hybrids(F_1,BC_1)of Saccharum×Erianthusrockii using morphology and GISH technique,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(6):2307-2316(林秀琴,陆鑫,刘新龙,毛钧,字秋艳,刘洪博,李忠磊,李旭娟,徐超华,李纯佳,蔡青,甘蔗与滇蔗茅杂交后代F_1、BC_1表型和GISH分析,分子植物育种,12(6):2307-2316
Liu L.,Du H.,Tang X.F.,Wu Y.M.,Huang Y.B.,and Tang Y.X.,2008,The roles of MYB transcription factors on plant defense responses and its molecu lar mechanism,Yichuan(Hereditas),30(10):1265-1271(刘蕾,杜海,唐晓凤,吴燕民,黄玉碧,唐益雄,2008,MYB转录因子在植物抗逆胁迫中的作用及其分子机理,遗传,30(10):1265-1271)
Lu X.,Mao J.,Liu H.B.,Liu X.L.,Ma L.,Su H.S.,and Cai Q.,2012,Innovation germplasm of Erianthus rockii establishment of F1intergeneric distant hy bridization of Saccharum officinarum and Erianthus rockii and the identification of F1by SSR molecular markers,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),13(2):321-324(陆鑫,毛钧,刘洪博,刘新龙,马丽,苏火生,蔡青,2012,甘蔗野生种滇蔗茅种质创新利用研究Ⅰ甘蔗与滇蔗茅远缘杂交F1群体构建与SSR分子标记鉴定,植物遗传资源学报,13(2):321-324)
Mortazavi A.,Williams B.A.,Mc Cue K.,Schaeffer L.,and Wold B.,2008,Mapping and quantifying mammalian transcriptomes by RNA-Seq,Nat.Methods,5(7):621-628
Niu J.Q.,Tan X.H.,Huang J.L.,Liu M.,Yang L.T.,Li Y.R.,and Wang A.Q.,2014,Effect of chilling stress on sucrose metabolism enzymes in leaf of sugarcane varieties with different cold resistance,Nanfang Nongye Xuebao(Journal of Southern Agriculture),45(9):1566-1573(牛俊奇,檀小辉,黄静丽,刘铭,杨丽涛,李杨瑞,王爱勤,2014,低温胁迫对不同耐寒型甘蔗蔗糖代谢相关酶基因表达及其酶活性的影响,南方农业学报,45(9):1566-1573)
Qiao X.Y.,Ma C.L.,and Chen L.,2009,Plant flavonoid biosynthesis pathway and regulation of its impor tant genes,Tianran Chanwu Yanjiu Yu Kaifa(Natural Product Research and Development),21(2):207-360(乔小燕,马春雷,陈亮,2009,植物类黄酮生物合成途径及重要基因的调控,天然产物研究与开发,21(2):207-360)
Qin Y.Z.,Xing Z.,Zhou J.F.,He C.Z.,Li Y.L.,and Xiong X.Y.,2014,Effects of sustained weak light on seedling growth and photosynthetic characteristics of potato seedlings,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),47(3):537-545(秦玉芝,邢铮,邹剑锋,何长征,李炎林,熊兴耀,2014,持续弱光胁迫对马铃薯苗期生长和光合特性的影响,中国农业科学,47(3):537-545)
Que Y.X.,Xu L.P.,Xu J.S.,Zhang J.S.,Zhang M.Q.,and Chen R.K.,2009,Selection of control genes in real-time q PCRanalysis of gene expression in sugarcane,R edai Zuowu Xuebao(Chinese Journal of Tropical Crops),30(3):274-278(阙有雄,许莉萍,徐景升,张积森,张木清,陈如凯,2009,甘蔗基因表达定量PCR分析中内参基因的选择,热带作物学报,30(3):274-278)
Sun A.Q.,Sun J.D.,Wan Y.S.,Liu F.Z.,Zhang K.,and Sun L.,2013,In silico expression profile of genes in response to drought in peanut,Zuowu Xuebao(Acta Agronomica Sinica),39(6):1045-1053(孙爱清,张杰道,万勇善,刘风珍,张昆,孙利,2013,花生干旱胁迫响应基因的数字表达谱分析,作物学报,39(6):1045-1053)
Wang L.P.,Fan Y.H.,Ma L.,Cai Q.,and Xia H.M.,1999,The research on photoperiod induction flowering of sugarcane and their utilization in hybridization,Ganzhe(Sugarcane),6(3):1-5(王丽萍,范源洪,马丽,蔡青,夏红明,1999,甘蔗光周期诱导开花和杂交利用研究,甘蔗,6(3):1-5)
Xu C.H.,Lu X.,Liu X.L.,Liu H.B.,Su H.S.,Ma L.,Mao J.,Lin X.Q.,Li X.J.,and Cai Q.,2014,Exploration and phenotypic traits analysis of Eri anthus fulvus,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),15(5):962-966(徐超华,陆鑫,刘新龙,刘洪博,苏火生,马丽,毛钧,林秀琴,李旭娟,蔡青,2014,甘蔗近缘种蔗茅Erianthus fulvus考察收集与表型性状初步研究,植物遗传资源学报,15(5):962-966)
Xu Y.,Chen J.H.,Zhu A.G.,Luan M.B.,Wang X.F.,and Sun Z.M.,2015,Research progress on re sponse mechanism of plant under low temperature stress,Zhongguo Maye Kexue(Plant Fiber Sciences in China),37(1):40-49(许英,陈建华,朱爱国,栾明宝,王晓飞,孙志民,2015,低温胁迫下植物响应机理的研究进展,中国麻业科学,37(1):40-49)
Yang H.J.,and Guo H.C.,2017,Digital gene expression profiling analysis of potato under low temperature stress,Zuowu Xuebao(Acta Agronomica Sinica),43(3):454-463(杨慧菊,郭华春,2017,低温胁迫下马铃薯的数字基因表达谱分析,作物学报,43(3):454-463)
Zhao J.L.,Wang X.H.,Li F.S.,He L.L.,Lou H.B.,Yang Q.H.,He S.C.,and Tang R.P.,2014,Genetic diversity of wild sugarcane species Erianthus fulvus based on SSR,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(2):323-331(赵锦龙,王先宏,李富生,何丽莲,娄红波,杨清辉,何顺长,唐荣平,2014,基于SSR标记的甘蔗野生种蔗茅遗传多样性分析,分子植物育种,12(2):323-331)
Zhao Y.C.,Liu X.W.,Li Y.,and Zhao D.G.,2016,Transcriptome data assembly and gene Function annotation of Zanthoxylum piperitum DC var.inerme makino,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(7):1805-1819(赵懿琛,刘雪微,李岩,赵德刚,2016,朝仓花椒幼芽转录组测序数据组装及基因功能注释,基因组学与应用生物学,35(7):1805-1819)
Bhardwaj A.R.,Joshi G.,Kukreja B.,Malik V.,Arora P.,Pandey R.,Shukla R.N.,Bankar K.G.,Agarwal S.K.,Goel S.,Jagannath A.,Kumar A.,and Agarwal M.,2015,Global insights into high temperature and drought stress regulated gen es by RNA-Seq in economically important oilseed crop Brassica juncea,BMC Plant Biol.,15(1):9
Fang Z.C.,Liu S.C.,Gao X.X.,Dao J.M.,and Fan X.,2014,Review on sugarcane stress physiology,Zhongguo Tangliao(Sugar Crops of China),39(4):79-82(方志存,刘少春,高欣欣,刀静梅,樊仙,2014,甘蔗逆境生理研究进展,中国糖料,39(4):79-82)
Hu H.L.,Hou X.M.,Qu B.,Gao X.J.,and Li Q.Z.,2012,Application of high throughput gene expression profiling,Zhongguo Ruping Gongye(China Dairy Industry),40(12):40-43(胡红柳,侯晓明,曲波,高学军,李庆章,2012,高通量基因表达谱的应用,中国乳品工业,40(12):40-43)
Huang X.,Chen M.H.,Yang L.T.,Zhang B.Q.,and Li Y.R.,2013,Effects of exogenous abscisic acid on cell membrane and endogenous hormones contents in leaves of sugarcane seedling under cold stress,Huazhong Nongye Daxue Xuebao(Journal of Huazhong Agricultural University),32(4):6-11(黄杏,陈明辉,杨丽涛,张保青,李杨瑞,2013,低温胁迫下外源ABA对甘蔗幼苗抗寒性及内源激素的影响,华中农业大学学报,32(4):6-11)
Li F.S.,Lin W.F.,and He S.C.,2004,Suggestions on developing and utilization of Erianthus fulvus wild species,Ziyuan Kaifa Yu Shichang(Resource Development&Market),20(4):266-270(李富生,林位夫,何顺长,2004,开发利用蔗茅野生种质资源的思考,资源开发与市场,20(4):266-270)
Li W.F.,Cai Q.,Huang Y.K.,Fan Y.H.,and Ma L.,2005,Identification of sugarcane wild germplasm resources resistant to Puccinia erianthi,Zhiwu Baohu(Plant Protection),31(2):51-53(李文凤,蔡青,黄应昆,范源洪,马丽,2005,甘蔗野生资源对蔗茅柄锈菌的抗性鉴定,植物保护,31(2):51-53)
Lin X.Q.,Liu X.,Liu X.L.,Mao J.,Zi Y.Q.,Liu H.B.,Li Z.L.,Li X.J.,Xu C.H.,Li C.J.,and Cai Q.,2016,Analysis of intergeneric hybrids(F_1,BC_1)of Saccharum×Erianthusrockii using morphology and GISH technique,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(6):2307-2316(林秀琴,陆鑫,刘新龙,毛钧,字秋艳,刘洪博,李忠磊,李旭娟,徐超华,李纯佳,蔡青,甘蔗与滇蔗茅杂交后代F_1、BC_1表型和GISH分析,分子植物育种,12(6):2307-2316
Liu L.,Du H.,Tang X.F.,Wu Y.M.,Huang Y.B.,and Tang Y.X.,2008,The roles of MYB transcription factors on plant defense responses and its molecu lar mechanism,Yichuan(Hereditas),30(10):1265-1271(刘蕾,杜海,唐晓凤,吴燕民,黄玉碧,唐益雄,2008,MYB转录因子在植物抗逆胁迫中的作用及其分子机理,遗传,30(10):1265-1271)
Lu X.,Mao J.,Liu H.B.,Liu X.L.,Ma L.,Su H.S.,and Cai Q.,2012,Innovation germplasm of Erianthus rockii establishment of F1intergeneric distant hy bridization of Saccharum officinarum and Erianthus rockii and the identification of F1by SSR molecular markers,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),13(2):321-324(陆鑫,毛钧,刘洪博,刘新龙,马丽,苏火生,蔡青,2012,甘蔗野生种滇蔗茅种质创新利用研究Ⅰ甘蔗与滇蔗茅远缘杂交F1群体构建与SSR分子标记鉴定,植物遗传资源学报,13(2):321-324)
Mortazavi A.,Williams B.A.,Mc Cue K.,Schaeffer L.,and Wold B.,2008,Mapping and quantifying mammalian transcriptomes by RNA-Seq,Nat.Methods,5(7):621-628
Niu J.Q.,Tan X.H.,Huang J.L.,Liu M.,Yang L.T.,Li Y.R.,and Wang A.Q.,2014,Effect of chilling stress on sucrose metabolism enzymes in leaf of sugarcane varieties with different cold resistance,Nanfang Nongye Xuebao(Journal of Southern Agriculture),45(9):1566-1573(牛俊奇,檀小辉,黄静丽,刘铭,杨丽涛,李杨瑞,王爱勤,2014,低温胁迫对不同耐寒型甘蔗蔗糖代谢相关酶基因表达及其酶活性的影响,南方农业学报,45(9):1566-1573)
Qiao X.Y.,Ma C.L.,and Chen L.,2009,Plant flavonoid biosynthesis pathway and regulation of its impor tant genes,Tianran Chanwu Yanjiu Yu Kaifa(Natural Product Research and Development),21(2):207-360(乔小燕,马春雷,陈亮,2009,植物类黄酮生物合成途径及重要基因的调控,天然产物研究与开发,21(2):207-360)
Qin Y.Z.,Xing Z.,Zhou J.F.,He C.Z.,Li Y.L.,and Xiong X.Y.,2014,Effects of sustained weak light on seedling growth and photosynthetic characteristics of potato seedlings,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),47(3):537-545(秦玉芝,邢铮,邹剑锋,何长征,李炎林,熊兴耀,2014,持续弱光胁迫对马铃薯苗期生长和光合特性的影响,中国农业科学,47(3):537-545)
Que Y.X.,Xu L.P.,Xu J.S.,Zhang J.S.,Zhang M.Q.,and Chen R.K.,2009,Selection of control genes in real-time q PCRanalysis of gene expression in sugarcane,R edai Zuowu Xuebao(Chinese Journal of Tropical Crops),30(3):274-278(阙有雄,许莉萍,徐景升,张积森,张木清,陈如凯,2009,甘蔗基因表达定量PCR分析中内参基因的选择,热带作物学报,30(3):274-278)
Sun A.Q.,Sun J.D.,Wan Y.S.,Liu F.Z.,Zhang K.,and Sun L.,2013,In silico expression profile of genes in response to drought in peanut,Zuowu Xuebao(Acta Agronomica Sinica),39(6):1045-1053(孙爱清,张杰道,万勇善,刘风珍,张昆,孙利,2013,花生干旱胁迫响应基因的数字表达谱分析,作物学报,39(6):1045-1053)
Wang L.P.,Fan Y.H.,Ma L.,Cai Q.,and Xia H.M.,1999,The research on photoperiod induction flowering of sugarcane and their utilization in hybridization,Ganzhe(Sugarcane),6(3):1-5(王丽萍,范源洪,马丽,蔡青,夏红明,1999,甘蔗光周期诱导开花和杂交利用研究,甘蔗,6(3):1-5)
Xu C.H.,Lu X.,Liu X.L.,Liu H.B.,Su H.S.,Ma L.,Mao J.,Lin X.Q.,Li X.J.,and Cai Q.,2014,Exploration and phenotypic traits analysis of Eri anthus fulvus,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),15(5):962-966(徐超华,陆鑫,刘新龙,刘洪博,苏火生,马丽,毛钧,林秀琴,李旭娟,蔡青,2014,甘蔗近缘种蔗茅Erianthus fulvus考察收集与表型性状初步研究,植物遗传资源学报,15(5):962-966)
Xu Y.,Chen J.H.,Zhu A.G.,Luan M.B.,Wang X.F.,and Sun Z.M.,2015,Research progress on re sponse mechanism of plant under low temperature stress,Zhongguo Maye Kexue(Plant Fiber Sciences in China),37(1):40-49(许英,陈建华,朱爱国,栾明宝,王晓飞,孙志民,2015,低温胁迫下植物响应机理的研究进展,中国麻业科学,37(1):40-49)
Yang H.J.,and Guo H.C.,2017,Digital gene expression profiling analysis of potato under low temperature stress,Zuowu Xuebao(Acta Agronomica Sinica),43(3):454-463(杨慧菊,郭华春,2017,低温胁迫下马铃薯的数字基因表达谱分析,作物学报,43(3):454-463)
Zhao J.L.,Wang X.H.,Li F.S.,He L.L.,Lou H.B.,Yang Q.H.,He S.C.,and Tang R.P.,2014,Genetic diversity of wild sugarcane species Erianthus fulvus based on SSR,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(2):323-331(赵锦龙,王先宏,李富生,何丽莲,娄红波,杨清辉,何顺长,唐荣平,2014,基于SSR标记的甘蔗野生种蔗茅遗传多样性分析,分子植物育种,12(2):323-331)
Zhao Y.C.,Liu X.W.,Li Y.,and Zhao D.G.,2016,Transcriptome data assembly and gene Function annotation of Zanthoxylum piperitum DC var.inerme makino,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(7):1805-1819(赵懿琛,刘雪微,李岩,赵德刚,2016,朝仓花椒幼芽转录组测序数据组装及基因功能注释,基因组学与应用生物学,35(7):1805-1819)