茶树CsCIGR基因克隆及表达特性分析
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摘要
该研究以茶树基因组数据库为基础,采用RT-PCR技术,从茶树‘龙井43’中克隆得到基因CsCIGR。序列分析显示,CsCIGR基因开放阅读框长度为1 677 bp,编码588个氨基酸。进化分析表明,CsCIGR属于GRAS家族的PAT1亚家族。多序列比对显示,茶树CsCIGR蛋白与其他植物的GRAS蛋白氨基酸序列具有很高的相似性。氨基酸理化性质分析显示,CsCIGR转录因子属于亲水性蛋白。亚细胞定位预测显示,CsCIGR可能位于细胞核中。启动子预测分析发现,CsCIGR启动子区域包含胁迫响应元件(STRE)、干旱应答元件(MYC)、厌氧诱导元件(ARE)等多种与逆境响应相关的顺式作用元件。荧光定量PCR分析结果显示,CsCIGR基因在低温(4℃)、高温(38℃)、干旱(200 g·L~(-1) PEG)、高盐(200 mmol·L~(-1) NaCl)胁迫下均能诱导表达,且对高盐,低温和高温胁迫响应更为明显,推测CsCIGR基因在茶树响应逆境胁迫中发挥重要作用。该研究为茶树抗性育种筛选基因提供了重要理论依据。
In this study, the CsCIGR gene was cloned from Camellia sinensis cultivar ‘Longjing 43' by RT-PCR method based on tea genome database. Sequence analysis showed that the open reading frame length of CsCIGR gene was 1677 bp, encoding 588 amino acids. Evolutionary analysis indicated that CsCIGR belongs to the PAT1 subfamily of the GRAS family. Multiple sequence alignments indicate that the CsCIGR protein of tea plants has a high degree of similarity to the amino acid sequences of GRAS proteins from other plants. The physicochemical properties of amino acids showed that CsCIGR transcription factor was a hydrophilic proteins. Subcellular localization prediction indicates that CsCIGR may be located in the nucleus. Promoter prediction analysis showed that the promoter region of CsCIGR contained stress response element(STRE), drought response element(MYC), anaerobic inducer(ARE) and other cis-acting elements related to stress response. The results of qRT-PCR showed that the CsCIGR gene responded to high temperature(38 ℃), low temperature(4 ℃), drought(200 g·L~(-1) PEG) and high salinity(200 mmol·L~(-1) NaCl). The expression levels of CsCIGR were significantly induced by high salinity, low temperature, and high temperature stresses. It is speculated that CsCIGR gene plays an important role in the response of C. sinensis to stresses. The results of this study provide important theoretical basis for screening genes of C. sinensis resistance breeding.
In this study, the CsCIGR gene was cloned from Camellia sinensis cultivar ‘Longjing 43' by RT-PCR method based on tea genome database. Sequence analysis showed that the open reading frame length of CsCIGR gene was 1677 bp, encoding 588 amino acids. Evolutionary analysis indicated that CsCIGR belongs to the PAT1 subfamily of the GRAS family. Multiple sequence alignments indicate that the CsCIGR protein of tea plants has a high degree of similarity to the amino acid sequences of GRAS proteins from other plants. The physicochemical properties of amino acids showed that CsCIGR transcription factor was a hydrophilic proteins. Subcellular localization prediction indicates that CsCIGR may be located in the nucleus. Promoter prediction analysis showed that the promoter region of CsCIGR contained stress response element(STRE), drought response element(MYC), anaerobic inducer(ARE) and other cis-acting elements related to stress response. The results of qRT-PCR showed that the CsCIGR gene responded to high temperature(38 ℃), low temperature(4 ℃), drought(200 g·L~(-1) PEG) and high salinity(200 mmol·L~(-1) NaCl). The expression levels of CsCIGR were significantly induced by high salinity, low temperature, and high temperature stresses. It is speculated that CsCIGR gene plays an important role in the response of C. sinensis to stresses. The results of this study provide important theoretical basis for screening genes of C. sinensis resistance breeding.
引文
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[12] 周莲洁,杨中敏,张富春,等.新疆盐穗木GRAS转录因子基因克隆及表达分析[J].西北植物学报,2013,33(6):1 091-1 097.ZHOU L J,YANG Z M,YANG F C,et al.Expression analysis and cloning of GRAS transcription factor gene from Halostachys caspica[J].Acta Botanica Boreali-Occidenta-lia Sinica,2013,33(6):1 091-1 097.
[13] 丁雪峰.水稻OsGRAS1启动子的克隆及多样性分析[J].上海农业学报,2010,26(4):8-14.DING X F.Cloning and diversity analysis of the OsGRAS1 promoter in rice[J].Acta Agriculturae Shanghai,2010,26(4):8-14.
[14] 曹丹,马林龙,金孝芳,等.茶树抗非生物逆境的研究进展[J].湖南农业科学,2015,(10):152-154.CAO D,MA L L,JIN X F,et al.Research advance of resistance to abiotic stresses of tea[J].Hunan Agriculturae Sciences,2015,(10):152-154.
[15] SHENG J,KIM C,ZHANG Y,et al.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis[J].Molecular Plant,2017,10(6):866-877.
[16] KUMAR S,STECHER G,TAMURA K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology & Evolution,2016,33(7):1 870-1 874.
[17] GOLDBERG T,HECHT M,HAMP T,et al.Loc Tree3 prediction of localization[J].Nucleic Acids Research,2014,42(Web Server issue):W350-W355.
[18] 沈威,滕瑞敏,李辉,等.茶树MADS-box转录因子基因的克隆与非生物胁迫响应分析[J].茶叶科学,2017,37(6):575-585.SHEN W,TENG R M,LI H,et al.Cloning of a MADS-box transcription factor gene from Camellia sinensis and its response to abiotic stresses[J].Journal of Tea Science,2017,37(6):575-585.
[19] 任燕,高童,陈江飞,等.茶树CsLEA5基因的克隆及表达分析[J].西北植物学报,2018,38(12):2 186-2 193.REN Y,GAO T,CHEN J F,et al.Cloning and expression analysis of CsLEA5 gene in tea plant (Camellia sinensis) [J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(12):2 186-2 193.
[20] COMBET C,BLANCHET C,GEOURJON C,et al.NPS@:network protein sequence analysis[J].Trends in Biochemical Sciences,2000,25(3):147-150.
[21] WU Z J,TIAN C,JIANG Q,et al.Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant (Camellia sinensis) [J].Scientific Reports,2016,6:19 748.
[22] PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45.
[23] WANG Y X,LIU Z W,WU Z J,et al.Genome-wide identification and expression analysis of GRAS family transcription factors in tea plant (Camellia sinensis) [J].Scientific Reports,2018,8(1):3 949.
[24] 段秋红.拟南芥中与GA20ox1基因启动子互作的转录因子筛选及功能初步研究[D].长沙:湖南大学,2014.
[25] 张玥,胡雲飞,王树茂,等.茶树儿茶素合成相关MYB转录因子生物信息学分析[J].茶叶科学,2018,38(2):162-173.ZHANG Y,HU Y F,WANG S M,et al.Bioinformatic analysis of MYB transcription factors involved in catechins biosynthesis in tea plant (Camellia sinensis) [J].Journal of Tea Science,2018,38(2):162-173.
[26] 韩雅彭,程琳,杨凌霄,等.茶树NAC转录因子家族的鉴定及生物信息学分析[J].河南大学学报(自然科学版),2017,47(3):301-309.HAN Y P,CHENG L,YANG L X,et al.Identification and bioinformatics analysis of NAC transcription factor family in tea tree (Camellia sinensis) [J].Journal of Henan University (Natural Science),2017,47(3):301-309.
[27] 贡年娣,郭丽丽,王弘雪,等.茶树两个MYB转录因子基因的克隆及功能验证[J].茶叶科学,2014,34(1):36-44.GONG N D,GUO L L,WANG H X,et al.Cloning and functional verification of two MYB transcription factors in tea plant [Camellia sinensis (L.)] [J].Journal of Tea Science,2014,34(1):36-44.
[28] 杨辉,杨晓娜,王翀,等.烟草NtGRAS基因的克隆与转录激活及表达特性分析[J].湖南农业大学学报(自然科学版),2018,44(3):271-277.YANG H,YANG X N,WANG C,et al.Clone of NtGRAS gene in tobacco and its transcriptional activation and expression analysis[J].Journal of Hunan Agricultural University (Natural Sciences),2018,44(3):271-277.
[29] 殷龙飞,张中保,于荣,等.植物GRAS家族蛋白结构和功能的研究进展[J/OL].分子植物育种:1-21[2019-03-03].http://kns.cnki.net/kcms/detail/46.1068.S.20190219.1612.006.html.YIN L F,ZHANG Z B,YU R,et al.Progress of the structural and functional analysis of GRAS gene in plants[J/OL].Molecular Plant Breeding:1-21[2019-03-03].http://kns.cnki.net/kcms/detail/46.1068.S.20190219.1612.006.html.
[30] 周莲洁,张富春,王艳.GRAS家族基因在植物生长、代谢及逆境胁迫中的功能研究进展[J].植物生理学报,2013,49(9):855-860.ZHOU L J,ZHANG F C,WANG Y.Research progress on the functional mechanism of GRAS family genes in plant growth,metabolism and stress[J].Plant Physiology Journal,2013,49(9):855-860.
[31] 粟莉圆,李孝哲,陈淑雯,等.核桃JrGRAS2基因响应热胁迫的表达及功能分析[J].植物研究,2018,38(1):125-131.SU L Y,LI X Z,CHEN S W,et al.Expression and function analysis of walnut JrGRAS2 gene under heat stress[J].Bulletin of Botanical Research,2018,38(1):125-131.
[2] YU Z,YAN L,WANG S,et al.Molecular cloning and characterization of galactinol synthases in Camellia sinensis with different responses to biotic and abiotic stressors[J].Journal of Agricultural & Food Chemistry,2017,65(13):2 751-2 759.
[3] ABARCA D,PIZARRO A,et al.The GRAS gene family in pine:transcript expression patterns associated with the maturation-related decline of competence to form adventitious roots[J].BMC Plant Biology,2014,14(1):354.
[4] 牛义岭,姜秀明,许向阳,等.植物转录因子GRAS蛋白的研究进展[J].基因组学与应用生物学,2016,35(9):2 519-2 524.NIU Y L,JIANG X M,XU X Y,et al.Research progress of transcription factors GRAS proteins in plant[J].Genomics and Applied Biology,2016,35(9):2 519-2 524.
[5] CHEN Y Q,TAI S S,WANG D W,et al.Homology-based analysis of the GRAS gene family in tobacco[J].Genetics & Molecular Research,2015,14(4):15 188-15 200.
[6] CENCI A,ROUARD M.Evolutionary analyses of GRAS transcription factors in Angiosperms[J].Frontiers in Plant Science,2017,8:273.
[7] FAN S,ZHANG D,GAO C,et al.Identification,classification,and expression analysis of GRAS gene family in Malus domestica[J].Frontiers in Physiology,2017,8:253.
[8] HAKOSHIMA T.Structural basis of the specific interactions of GRAS family proteins[J].FEBS Letters,2018,592(4):489-501.
[9] 郭玉玉.玉米GRAS转录因子家族基因克隆与功能验证[D].山东泰安:山东农业大学,2015.
[10] 郭华军,焦远年,邸超,等.拟南芥转录因子GRAS家族基因群响应渗透和干旱胁迫的初步探索[J].植物学报,2009,44(3):290-299.GUO H J,JIAO Y N,DI C,et al.Preliminary study on response to infiltration and drought stress of Arabidopsis thaliana transcription factor GRAS family gene group[J].Chinese Bulletin of Botany,2009,44(3):290-299.
[11] 石瑞,曹诣斌,陈文荣,等.佛手GRAS基因的克隆及表达分析[J].浙江师范大学学报(自然科学版),2011,34(4):446-451.SHI R,CAO Y B,CHEN W R,et al.On cDNA cloning and expression analysis of GRAS gene in fingered citron[J].Journal of Zhejiang Normal University (Nat.Sci.),2011,34(4):446-451.
[12] 周莲洁,杨中敏,张富春,等.新疆盐穗木GRAS转录因子基因克隆及表达分析[J].西北植物学报,2013,33(6):1 091-1 097.ZHOU L J,YANG Z M,YANG F C,et al.Expression analysis and cloning of GRAS transcription factor gene from Halostachys caspica[J].Acta Botanica Boreali-Occidenta-lia Sinica,2013,33(6):1 091-1 097.
[13] 丁雪峰.水稻OsGRAS1启动子的克隆及多样性分析[J].上海农业学报,2010,26(4):8-14.DING X F.Cloning and diversity analysis of the OsGRAS1 promoter in rice[J].Acta Agriculturae Shanghai,2010,26(4):8-14.
[14] 曹丹,马林龙,金孝芳,等.茶树抗非生物逆境的研究进展[J].湖南农业科学,2015,(10):152-154.CAO D,MA L L,JIN X F,et al.Research advance of resistance to abiotic stresses of tea[J].Hunan Agriculturae Sciences,2015,(10):152-154.
[15] SHENG J,KIM C,ZHANG Y,et al.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis[J].Molecular Plant,2017,10(6):866-877.
[16] KUMAR S,STECHER G,TAMURA K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology & Evolution,2016,33(7):1 870-1 874.
[17] GOLDBERG T,HECHT M,HAMP T,et al.Loc Tree3 prediction of localization[J].Nucleic Acids Research,2014,42(Web Server issue):W350-W355.
[18] 沈威,滕瑞敏,李辉,等.茶树MADS-box转录因子基因的克隆与非生物胁迫响应分析[J].茶叶科学,2017,37(6):575-585.SHEN W,TENG R M,LI H,et al.Cloning of a MADS-box transcription factor gene from Camellia sinensis and its response to abiotic stresses[J].Journal of Tea Science,2017,37(6):575-585.
[19] 任燕,高童,陈江飞,等.茶树CsLEA5基因的克隆及表达分析[J].西北植物学报,2018,38(12):2 186-2 193.REN Y,GAO T,CHEN J F,et al.Cloning and expression analysis of CsLEA5 gene in tea plant (Camellia sinensis) [J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(12):2 186-2 193.
[20] COMBET C,BLANCHET C,GEOURJON C,et al.NPS@:network protein sequence analysis[J].Trends in Biochemical Sciences,2000,25(3):147-150.
[21] WU Z J,TIAN C,JIANG Q,et al.Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant (Camellia sinensis) [J].Scientific Reports,2016,6:19 748.
[22] PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45.
[23] WANG Y X,LIU Z W,WU Z J,et al.Genome-wide identification and expression analysis of GRAS family transcription factors in tea plant (Camellia sinensis) [J].Scientific Reports,2018,8(1):3 949.
[24] 段秋红.拟南芥中与GA20ox1基因启动子互作的转录因子筛选及功能初步研究[D].长沙:湖南大学,2014.
[25] 张玥,胡雲飞,王树茂,等.茶树儿茶素合成相关MYB转录因子生物信息学分析[J].茶叶科学,2018,38(2):162-173.ZHANG Y,HU Y F,WANG S M,et al.Bioinformatic analysis of MYB transcription factors involved in catechins biosynthesis in tea plant (Camellia sinensis) [J].Journal of Tea Science,2018,38(2):162-173.
[26] 韩雅彭,程琳,杨凌霄,等.茶树NAC转录因子家族的鉴定及生物信息学分析[J].河南大学学报(自然科学版),2017,47(3):301-309.HAN Y P,CHENG L,YANG L X,et al.Identification and bioinformatics analysis of NAC transcription factor family in tea tree (Camellia sinensis) [J].Journal of Henan University (Natural Science),2017,47(3):301-309.
[27] 贡年娣,郭丽丽,王弘雪,等.茶树两个MYB转录因子基因的克隆及功能验证[J].茶叶科学,2014,34(1):36-44.GONG N D,GUO L L,WANG H X,et al.Cloning and functional verification of two MYB transcription factors in tea plant [Camellia sinensis (L.)] [J].Journal of Tea Science,2014,34(1):36-44.
[28] 杨辉,杨晓娜,王翀,等.烟草NtGRAS基因的克隆与转录激活及表达特性分析[J].湖南农业大学学报(自然科学版),2018,44(3):271-277.YANG H,YANG X N,WANG C,et al.Clone of NtGRAS gene in tobacco and its transcriptional activation and expression analysis[J].Journal of Hunan Agricultural University (Natural Sciences),2018,44(3):271-277.
[29] 殷龙飞,张中保,于荣,等.植物GRAS家族蛋白结构和功能的研究进展[J/OL].分子植物育种:1-21[2019-03-03].http://kns.cnki.net/kcms/detail/46.1068.S.20190219.1612.006.html.YIN L F,ZHANG Z B,YU R,et al.Progress of the structural and functional analysis of GRAS gene in plants[J/OL].Molecular Plant Breeding:1-21[2019-03-03].http://kns.cnki.net/kcms/detail/46.1068.S.20190219.1612.006.html.
[30] 周莲洁,张富春,王艳.GRAS家族基因在植物生长、代谢及逆境胁迫中的功能研究进展[J].植物生理学报,2013,49(9):855-860.ZHOU L J,ZHANG F C,WANG Y.Research progress on the functional mechanism of GRAS family genes in plant growth,metabolism and stress[J].Plant Physiology Journal,2013,49(9):855-860.
[31] 粟莉圆,李孝哲,陈淑雯,等.核桃JrGRAS2基因响应热胁迫的表达及功能分析[J].植物研究,2018,38(1):125-131.SU L Y,LI X Z,CHEN S W,et al.Expression and function analysis of walnut JrGRAS2 gene under heat stress[J].Bulletin of Botanical Research,2018,38(1):125-131.