黄金茶CsDAM2基因克隆及其功能分析
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摘要
休眠是通过植物体内相应基因的表达调控机制,使植物芽停止生长,以适应外界低温、短日照等不良环境条件的结果。茶芽打破休眠的早晚,密切关系到茶树经济生产的效益。为进一步掌握茶树芽休眠与解除的分子机理,本研究克隆了可能与黄金茶发芽早相关的转录因子(dormancy associated MADS-box Cs DAM2),并在烟草中过表达CsDAM2,对CsDAM2转基因烟草和野生型烟草进行低温处理。结果表明,在相同处理条件下,随着温度的降低,同野生型烟草比较,转基因烟草电导率增加幅度小,并且叶绿素荧光值降低率低,说明在抗寒性能上,转基因烟草具有显著优势,进而一定程度上证实了Cs DAM2具有提高茶树抗寒性的作用。本研究结果为进一步揭示茶树芽休眠与解除的分子机理提供了科学依据。
Do rmancy is the result of stopping the growth of plant buds by regulating the expression of corresponding genes in plants to adapt to the adverse environmental conditions such as low temperature and short sunshine. The time of tea buds breaking dormancy is closely related to the economic benefits of tea production. In order to further understand the molecular mechanism of tea bud dormancy and breaking, we cloned a transcription factor Dormancy Associated MADS-box(CsDAM2), which might be related to early germination of Huangjin tea,and overexpressed CsDAM2 in tobacco. The transgenic tobacco and wild-type tobacco were treated with low temperature. The results showed that under the same treatment conditions, with the decrease of temperature, the conductivity of transgenic tobacco increased slightly and the chlorophyll fluorescence value decreased slightly compared with wild-type tobacco. This indicated that transgenic tobacco had significant advantages in cold resistance, and further confirmed to some extent that CsDAM2 could improve cold resistance of tea plants. The results of this study could provide a scientific basis for further revealing the molecular mechanism of dormancy and breaking of tea buds.
Do rmancy is the result of stopping the growth of plant buds by regulating the expression of corresponding genes in plants to adapt to the adverse environmental conditions such as low temperature and short sunshine. The time of tea buds breaking dormancy is closely related to the economic benefits of tea production. In order to further understand the molecular mechanism of tea bud dormancy and breaking, we cloned a transcription factor Dormancy Associated MADS-box(CsDAM2), which might be related to early germination of Huangjin tea,and overexpressed CsDAM2 in tobacco. The transgenic tobacco and wild-type tobacco were treated with low temperature. The results showed that under the same treatment conditions, with the decrease of temperature, the conductivity of transgenic tobacco increased slightly and the chlorophyll fluorescence value decreased slightly compared with wild-type tobacco. This indicated that transgenic tobacco had significant advantages in cold resistance, and further confirmed to some extent that CsDAM2 could improve cold resistance of tea plants. The results of this study could provide a scientific basis for further revealing the molecular mechanism of dormancy and breaking of tea buds.
引文
Barua D.N.,1969,Seasonal dormancy in tea(Camellia sinensis L.),Nature,224(1):514
Chang X.,Pang L.,Li Y.Y.,Wei Y.L.,and Jiang C.J.,2012,Eukaryotic expression and cold resistance analysis of CsCBF1from tea plant(Camellia sinensis),Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest A&F University(Natural Science Edition)),40(10):173-179(常欣,庞磊,李叶云,魏艳丽,江昌俊,2012,茶树CsCBF1基因表达载体的构建及其转基因烟草的抗寒性分析,西北农林科技大学学报(自然科学版),40(10):173-179)
Chen L.B.,Li Y.Y.,Fang C.,Zhu Z.,and Jiang C.J.,2011,Screening genes related to cold stress from tea plant using AFLP,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),31(1):1-7(陈林波,李叶云,房超,朱政,江昌俊,2011,茶树冷诱导基因的AFLP筛选及其表达分析,西北植物学报,31(1):1-7)
Chen X.,Fang W.P.,Zou Z.W.,Wang Y.H.,Cheng H.,and Li X.H.,2009,Cloning and expression analysis of CBF gene in cold induced tea plant[Camellia Sinensis(L.)O.Kuntze],Chaye Kexue(Journal of Tea Science),29(1):53-59(陈暄,房婉萍,邹中伟,王玉花,成浩,黎星辉,2009,茶树冷胁迫诱导抗寒基因CBF的克隆与表达分析,茶叶科学,29(1):53-59)
Gu H.Y.,and Yuan X.W.,2010,A highly effective heat shock PCR screening method for recombination clone,Shengwuxue Zazhi(Journal of Biology),27(2):36-39(顾洪雁,袁晓伟,2010,一种热激PCR法高效鉴定重组克隆,生物学杂志,27(2):36-39)
Hoenicka H.,Nowitzki O.,Hanelt D.,and Fladung M.,2008,Heterologous overexpression of the birch FRUITFULL-like MADS-box gene BpMADS4 prevents normal senescence and winter dormancy in Populus tremula L.,Planta,227(5):1001-1011
Jiménez S.,Reighard G.L.,and Bielenberg D.G.,2010,Gene expression of DAM5 and DAM6 is suppressed by chilling temperatures and inversely correlated with bud break rate,Plant Mol.Biol.,73(1-2):157-167
Liu G.Q.,Zheng P.H.,Chen L.,and Teng Y.W.,2012,Research progresses on genes associated with bud dormancy in deciduous fruit trees,Guoshu Xuebao(Journal of Fruit Science),29(5):911-917(刘国琴,郑鹏华,滕元文,2012,落叶果树芽休眠相关基因的研究进展,果树学报,29(5):911-917)
Liu Z.,Yang Y.,Zhao Y.,Yang P.D.,Cheng Y.,and Long C.X.,2016,The breeding of early budding,high-yield and high quality new green tea cultivar Huangjincha 168,Chaye Tongxun(Journal of Tea Communication),43(2):13-18(刘振,杨阳,赵洋,杨培迪,成杨,龙承先,2016,特早生高产优质绿茶新品种黄金茶168号选育研究,茶叶通讯,43(2):13-18)
Liu Z.W.,Wu Z.J.,Li X.H.,Li T.,and Zhuang J.,2014,Gene cloning of CsDREB-A1 transcription factor from Camellia sinensis and its characteristic analysis,Zhiwu Ziyuan Yu Huanjing Xuebao(Journal of Plant Resources and Environment),23(4):8-16(刘志薇,吴致君,黎星辉,李彤,庄静,2014,茶树CsDREB-A1转录因子基因的克隆及其特性分析,植物资源与环境学报,23(4):8-16)
Ma Y.,Zhang H.X.,Tu Y.,and Chen X.L.,2017,Advances in research on transcription factors related to fruit development and ripening,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(11):4836-4846(马勇,张红霞,图雅,陈秀莉,2017,植物果实发育成熟相关转录因子的研究进展,基因组学与应用生物学,36(11):4836-4846)
Pang L.,Zhou X.S.,Li Y.Y.,and Jiang J.C.,2011,Study and identification of cold resistance of Camellia sinensis by chlorophyll fluorescence method,Chaye Kexue(Journal of Tea Science),31(6):521-524(庞磊,周小生,李叶云,江昌俊,2011,应用叶绿素荧光法鉴定茶树品种抗寒性的研究,茶叶科学,31(6):521-524)
Thirugnanasambantham K.,Prabu G.,Palanisamy S.,Chandrabose S.R.,and Mandal A.K.,2013,Analysis of dormant bud(Banjhi)specific transcriptome of tea(Camellia sinensis(L.)O.Kuntze)from cDNA library revealed dormancy-related genes,Applied Biochemistry and Biotechnology,169(4):1405-1417
Wang X.C.,Yang Y.J.,Chen L.,Ma C.L.,and Yao M.Z.,2010,Construction and preli minary analysis of the suppression subtractive hybridization cDNA libraries between dormant and sprouting buds of tea plant(Camellia sinensis),Chaye Kexue(Journal of Tea Science),30(2):129-135(王新超,杨亚军,陈亮,马春雷,姚明哲,2010,茶树休眠芽与萌动芽抑制消减杂交文库的构建与初步分析,茶叶科学,30(2):129-135)
Wang Y.,Jiang C.J.,Li Y.Y.,Wei C.L.,and Deng W.W.,2012,Cs ICE1 and Cs CBF1:two transcription factors involved in cold responses in Camellia sinensis,Plant Cell Reports,31(1):27-34
Yamane H.,Ooka T.,Jotatsu H.,Hosaka Y.,Sasaki R.,and Tao R.,2011,Expressional regulation of PpDAM5 and Pp DAM6,peach(Prunus persica)dormancy-associated MADS-box genes,by low temperature and dormancy-breaking reagent treatment,J.Exp.Bot.,62(10):3481-3488
Yang Y.J.,Zheng L.Y.,and Wang X.C.,2005,Changes of membrane fatty acid composition and protein in tea leaves at low temperature,Yaredai Zhiwu Kexue(Subtropical Plant Science),34(1):5-9(杨亚军,郑雷英,王新超,2005,低温对茶树叶片膜脂脂肪酸和蛋白质的影响,亚热带植物科学,34(1):5-9)
Zhang J.L.,Zhang L.X.,Yang K.M.,and Li Z.Y.,2017,A comparison study on extraction methods for genomic DNA from tissues of camellia plants with modified CTAB method,Jiangxi Hongye Daxue Xuebao(Acta Agriculturae Universitatis Jiangxiensis),39(4):785-791(张金丽,张罗霞,杨坤梅,李宗艳,2017,改良CTAB法对山茶属植物基因组DNA提取的比较研究,江西农业大学学报,39(4):785-791)
Zhang X.S.,2006,The present situation and prospect of utilization of local rare Tea variety--Golden tea,Chaye Tongxun(Tea Communication),33(2):4-6(张湘生,2006,地方珍稀茶树良种-黄金茶的利用现状与前景,茶叶通讯,33(2):4-6)
Zhao D.,Liu Z.S.,Lu J.L.,Qian L.S.,Tu Y.Y.,and Xi B.,2001,Study on agrobacterium tumefaciens-mediated transformation of tea plant,Chaye Kexue(Journal of Tea Science),21(2):108-111(赵东,刘祖生,陆建良,钱利生,屠幼英,奚彪,2001,根癌农杆菌介导茶树转化研究,茶叶科学,21(2):108-111)
Zhu Z.,Jiang J.Y.,Jiang C.J.,and Li W.,2011,Effects of low temperature stress on SOD activity,soluble protein content and soluble sugar content in Camellia sinensis leaves,Anhui Hongye Daxue Xuebao(Journal of Anhui Agricultural University),38(1):24-26(朱政,蒋家月,江昌俊,李雯,2011,低温胁迫对茶树叶片SOD,可溶性蛋白和可溶性糖含量的影响,安徽农业大学学报,38(1):24-26)
Zou Z.W.,Fang W.P.,Zhang D.,Duan Y.S.,and Li X.H.,2008,Analysis of differential expression genes in cold-induced tea plant,Chaye Kexue(Journal of Tea Science),28(4):249-254(邹中伟,房婉萍,张定,段云裳,黎星辉,2008,低温胁迫下茶树基因表达的差异分析,茶叶科学,28(4):249-254)
Chang X.,Pang L.,Li Y.Y.,Wei Y.L.,and Jiang C.J.,2012,Eukaryotic expression and cold resistance analysis of CsCBF1from tea plant(Camellia sinensis),Xibei Nonglin Keji Daxue Xuebao(Journal of Northwest A&F University(Natural Science Edition)),40(10):173-179(常欣,庞磊,李叶云,魏艳丽,江昌俊,2012,茶树CsCBF1基因表达载体的构建及其转基因烟草的抗寒性分析,西北农林科技大学学报(自然科学版),40(10):173-179)
Chen L.B.,Li Y.Y.,Fang C.,Zhu Z.,and Jiang C.J.,2011,Screening genes related to cold stress from tea plant using AFLP,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),31(1):1-7(陈林波,李叶云,房超,朱政,江昌俊,2011,茶树冷诱导基因的AFLP筛选及其表达分析,西北植物学报,31(1):1-7)
Chen X.,Fang W.P.,Zou Z.W.,Wang Y.H.,Cheng H.,and Li X.H.,2009,Cloning and expression analysis of CBF gene in cold induced tea plant[Camellia Sinensis(L.)O.Kuntze],Chaye Kexue(Journal of Tea Science),29(1):53-59(陈暄,房婉萍,邹中伟,王玉花,成浩,黎星辉,2009,茶树冷胁迫诱导抗寒基因CBF的克隆与表达分析,茶叶科学,29(1):53-59)
Gu H.Y.,and Yuan X.W.,2010,A highly effective heat shock PCR screening method for recombination clone,Shengwuxue Zazhi(Journal of Biology),27(2):36-39(顾洪雁,袁晓伟,2010,一种热激PCR法高效鉴定重组克隆,生物学杂志,27(2):36-39)
Hoenicka H.,Nowitzki O.,Hanelt D.,and Fladung M.,2008,Heterologous overexpression of the birch FRUITFULL-like MADS-box gene BpMADS4 prevents normal senescence and winter dormancy in Populus tremula L.,Planta,227(5):1001-1011
Jiménez S.,Reighard G.L.,and Bielenberg D.G.,2010,Gene expression of DAM5 and DAM6 is suppressed by chilling temperatures and inversely correlated with bud break rate,Plant Mol.Biol.,73(1-2):157-167
Liu G.Q.,Zheng P.H.,Chen L.,and Teng Y.W.,2012,Research progresses on genes associated with bud dormancy in deciduous fruit trees,Guoshu Xuebao(Journal of Fruit Science),29(5):911-917(刘国琴,郑鹏华,滕元文,2012,落叶果树芽休眠相关基因的研究进展,果树学报,29(5):911-917)
Liu Z.,Yang Y.,Zhao Y.,Yang P.D.,Cheng Y.,and Long C.X.,2016,The breeding of early budding,high-yield and high quality new green tea cultivar Huangjincha 168,Chaye Tongxun(Journal of Tea Communication),43(2):13-18(刘振,杨阳,赵洋,杨培迪,成杨,龙承先,2016,特早生高产优质绿茶新品种黄金茶168号选育研究,茶叶通讯,43(2):13-18)
Liu Z.W.,Wu Z.J.,Li X.H.,Li T.,and Zhuang J.,2014,Gene cloning of CsDREB-A1 transcription factor from Camellia sinensis and its characteristic analysis,Zhiwu Ziyuan Yu Huanjing Xuebao(Journal of Plant Resources and Environment),23(4):8-16(刘志薇,吴致君,黎星辉,李彤,庄静,2014,茶树CsDREB-A1转录因子基因的克隆及其特性分析,植物资源与环境学报,23(4):8-16)
Ma Y.,Zhang H.X.,Tu Y.,and Chen X.L.,2017,Advances in research on transcription factors related to fruit development and ripening,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(11):4836-4846(马勇,张红霞,图雅,陈秀莉,2017,植物果实发育成熟相关转录因子的研究进展,基因组学与应用生物学,36(11):4836-4846)
Pang L.,Zhou X.S.,Li Y.Y.,and Jiang J.C.,2011,Study and identification of cold resistance of Camellia sinensis by chlorophyll fluorescence method,Chaye Kexue(Journal of Tea Science),31(6):521-524(庞磊,周小生,李叶云,江昌俊,2011,应用叶绿素荧光法鉴定茶树品种抗寒性的研究,茶叶科学,31(6):521-524)
Thirugnanasambantham K.,Prabu G.,Palanisamy S.,Chandrabose S.R.,and Mandal A.K.,2013,Analysis of dormant bud(Banjhi)specific transcriptome of tea(Camellia sinensis(L.)O.Kuntze)from cDNA library revealed dormancy-related genes,Applied Biochemistry and Biotechnology,169(4):1405-1417
Wang X.C.,Yang Y.J.,Chen L.,Ma C.L.,and Yao M.Z.,2010,Construction and preli minary analysis of the suppression subtractive hybridization cDNA libraries between dormant and sprouting buds of tea plant(Camellia sinensis),Chaye Kexue(Journal of Tea Science),30(2):129-135(王新超,杨亚军,陈亮,马春雷,姚明哲,2010,茶树休眠芽与萌动芽抑制消减杂交文库的构建与初步分析,茶叶科学,30(2):129-135)
Wang Y.,Jiang C.J.,Li Y.Y.,Wei C.L.,and Deng W.W.,2012,Cs ICE1 and Cs CBF1:two transcription factors involved in cold responses in Camellia sinensis,Plant Cell Reports,31(1):27-34
Yamane H.,Ooka T.,Jotatsu H.,Hosaka Y.,Sasaki R.,and Tao R.,2011,Expressional regulation of PpDAM5 and Pp DAM6,peach(Prunus persica)dormancy-associated MADS-box genes,by low temperature and dormancy-breaking reagent treatment,J.Exp.Bot.,62(10):3481-3488
Yang Y.J.,Zheng L.Y.,and Wang X.C.,2005,Changes of membrane fatty acid composition and protein in tea leaves at low temperature,Yaredai Zhiwu Kexue(Subtropical Plant Science),34(1):5-9(杨亚军,郑雷英,王新超,2005,低温对茶树叶片膜脂脂肪酸和蛋白质的影响,亚热带植物科学,34(1):5-9)
Zhang J.L.,Zhang L.X.,Yang K.M.,and Li Z.Y.,2017,A comparison study on extraction methods for genomic DNA from tissues of camellia plants with modified CTAB method,Jiangxi Hongye Daxue Xuebao(Acta Agriculturae Universitatis Jiangxiensis),39(4):785-791(张金丽,张罗霞,杨坤梅,李宗艳,2017,改良CTAB法对山茶属植物基因组DNA提取的比较研究,江西农业大学学报,39(4):785-791)
Zhang X.S.,2006,The present situation and prospect of utilization of local rare Tea variety--Golden tea,Chaye Tongxun(Tea Communication),33(2):4-6(张湘生,2006,地方珍稀茶树良种-黄金茶的利用现状与前景,茶叶通讯,33(2):4-6)
Zhao D.,Liu Z.S.,Lu J.L.,Qian L.S.,Tu Y.Y.,and Xi B.,2001,Study on agrobacterium tumefaciens-mediated transformation of tea plant,Chaye Kexue(Journal of Tea Science),21(2):108-111(赵东,刘祖生,陆建良,钱利生,屠幼英,奚彪,2001,根癌农杆菌介导茶树转化研究,茶叶科学,21(2):108-111)
Zhu Z.,Jiang J.Y.,Jiang C.J.,and Li W.,2011,Effects of low temperature stress on SOD activity,soluble protein content and soluble sugar content in Camellia sinensis leaves,Anhui Hongye Daxue Xuebao(Journal of Anhui Agricultural University),38(1):24-26(朱政,蒋家月,江昌俊,李雯,2011,低温胁迫对茶树叶片SOD,可溶性蛋白和可溶性糖含量的影响,安徽农业大学学报,38(1):24-26)
Zou Z.W.,Fang W.P.,Zhang D.,Duan Y.S.,and Li X.H.,2008,Analysis of differential expression genes in cold-induced tea plant,Chaye Kexue(Journal of Tea Science),28(4):249-254(邹中伟,房婉萍,张定,段云裳,黎星辉,2008,低温胁迫下茶树基因表达的差异分析,茶叶科学,28(4):249-254)