根癌农杆菌介导地被菊‘紫妍’遗传转化体系的建立
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摘要
以地被菊‘紫妍’高效再生体系为基础,研究农杆菌介导的地被菊‘紫妍’遗传转化的若干影响因素。结果表明:卡那霉素6、4mg·L-1分别是叶片分化和植株生根的最适筛选质量浓度,同时确定羧苄霉素200mg·L-1是抑制农杆菌生长的最适质量浓度;预培养2d,侵染时菌液OD600约为0.5,侵染时间8min,共培养2d,延迟培养3d有利于提高转化频率。对抗性芽进行生根筛选后,抗性苗提取DNA经过PCR检测初步证明外源基因ClCBF4已整合到地被菊中。移栽出的转基因地被菊全部存活且生长状况良好。
Several factors,affecting Agrobacterium-mediated transformation of Chrysanthemum grandiflorum ‘Ziyan'were studied based on the establishment of high efficient regeneration system.The results showed that the most suitable concentration of Km for leaf differentiation and plant rooting is 4mg·L~(-1) and 6 mg·L~(-1).The most suitable concentration of Carb for inhibiting the growth of Agrobacterium tumefaciens is 200mg·L~(-1).The pre-culture time of 2days and Co-culture for 2days were beneficial for improving transformation frequency.The 8 min bacteria infection(OD600=0.5)with delay-culture of 3days were found to be optimal selection for transgenic plantlets.It was confirmed by using gene PCR test that the vector was integrated into the regeneration genome of Chrysanthemum grandiflorum ‘Ziyan'and the transgenic Chrysanthemum grandiflorum which were transplanted survived and grew well.
Several factors,affecting Agrobacterium-mediated transformation of Chrysanthemum grandiflorum ‘Ziyan'were studied based on the establishment of high efficient regeneration system.The results showed that the most suitable concentration of Km for leaf differentiation and plant rooting is 4mg·L~(-1) and 6 mg·L~(-1).The most suitable concentration of Carb for inhibiting the growth of Agrobacterium tumefaciens is 200mg·L~(-1).The pre-culture time of 2days and Co-culture for 2days were beneficial for improving transformation frequency.The 8 min bacteria infection(OD600=0.5)with delay-culture of 3days were found to be optimal selection for transgenic plantlets.It was confirmed by using gene PCR test that the vector was integrated into the regeneration genome of Chrysanthemum grandiflorum ‘Ziyan'and the transgenic Chrysanthemum grandiflorum which were transplanted survived and grew well.
引文
[1] 戴思兰,王文奎,黄家平.菊属系统学与菊花起源研究进展[J].北京林业大学学报,2002,24(5/6):230-234.DAI S L,WANG W K,HUANG J P.Advances of researches on phylogeny of Dendranthemaand origin of Chrysanthemum[J].Journal of Beijing Forestry Unicersity,2002,24(5/6):230-234(in Chinese with English abstract).
[2] 陈发棣,房伟民,赵宏波,等.菊花新品种-地被菊系列[J].园艺学报,2005(6):1167-1168.CHEN F L,FANG W M,ZHAO H B,et al.New varieties of Chrysanthemum-Ground Cover Varieties[J].Acta Horticulturae Sinica,2005(6):1167-1168(in Chinese with English abstract).
[3] 张晓娇,史春凤,李春水,等.转AtDREB1A基因地被菊杂交后代优株耐寒性分析[J].园艺学报,2011,38(9):1717-1726.ZHANG X J,SHI CH F,LI CH SH,et al.Cold stress tolerance of the filial generations produced by AtDREB1Atransgenic ground cover Chrysanthemumand a conventional variety[J].Acta Horticulturae Sinica,2011,38(9):1717-1726(in Chinese with English abstract).
[4] MORRAN S,EINI O,PYVOVARENKO T,et al.Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors[J].Plant Biotechnol Journal,2011,9(2):230-249.
[5] 翟俊峰,王法微,王 南,等.月季CBF转录因子基因的克隆及表达分析[J].园艺学报,2012,39(8):1596-1602.ZHAI J F,WANG F W,WANG N,et al.Cloning and expression profiling of the transcription factor CBFgene from rosa hybrida[J].Acta Horticulturae Sinica,2012,39(8):1596-1602(in Chinese with English abstract).
[6] SHAN W,KUANG J F,LU W J,et al.Banana fruit NAC transcription factor MaNACl is a direct target of MaICE1and involved in cold stress through interacting with MaCBF1[J].Plant Cell &Environment,2014.37(9):2116-2127.
[7] CHEN M,WANG Q Y,CHENG X G,et al.GmDREB2,a soybean DRE-Binding transcription factor,conferred drought and high-salt tolerance in transgenic plants[J].Biochemical and Biophysical Research Communications,2007,353(2):299-305.
[8] ZHOU M Q,SHEN C,WU L H,et al.CBF-dependent signaling pathway:a key responder to low temperature stress in plants[J].Critical Reviews in Biotechnology,2011,31(2):186-192.
[9] 刘 杰.甘菊ClCBF家族基因分离及其对拟南芥的转化[D].哈尔滨:东北林业大学,2013.LIU J.Isolation of CBFfamily genes fromChrysanthemum lavandulifolium and transformation studies of ClCBFin Arabidopsis thaliana[D].Harbin:Northeast Forestry University,2013(in Chinese with English abstract).
[10] 肖 政,范崇辉,金万梅.根癌农杆菌介导转录因子CBF1基因对菊花的转化[J].西北农业学报,2009,18(6):262-267.XIAO ZH,FAN CH H,JIN W M.Agrobacterium-mediated transformation of Deranthema morifolium Tzvel.cv.Xiaojinhuang with transcription factor CBF1 gene[J]Acta Agriculturae Boreali-Occidentalis Sinica,2009,18(6):262-267(in Chinese with English abstract).
[11] 向太和,王 琳,蒋 欢,等.发根农杆菌K599对菊花活体转化及其高效再生[J].园艺学报,2011,38(7):1365-1370.XIANG T H,WANG L,JIANG H,et al.Hairy roots induced by Agrobacterium thizogenes K599in Chrysanthemumin vivo and plant regeneration from hairy roots[J].Acta Horticulturae Sinica,2011,38(7):1365-1370(in Chinese with English abstract).
[12] 储 俊,许 娜,张 强,等.农杆菌介导的药百合鳞片遗传转化体系的建立[J].草业学报,2011,20(6):164-169.CHU J,XU N,ZHANG Q,et al.Establishment of an Agrobacterium tumefaciens-mediated genetic transformation system of Lilium speciosum[J].Acta Prataculturae Sinica,2011,20(6):164-169(in Chinese with English abstract).
[13] SUN H J,UCHII S,WATANABE S,et al.A highly efficient transformation protocol for Micro-Tom,a model cultivar for tomato functional genomics[J].Plant Cell Physiol,2006,47(3):426-431.
[14] KNIGHT M R,KNIGHT H.Low-temperature perception leading to gene expression and cold tolerance in higher plants[J].New Phytologist,2012,195(4):737-751.
[15] 霍秀文,米福贵,云锦凤,等.转录因子CBF4诱导型启动子的克隆及功能分析[J].分子植物育种,2005,3(3):363-368.HUO X W,MI F G,YUN J F,et al.Clonging and functional analysis of an induced promoter of transcriptional factor CBF4 from Arabidopsis[J].Molecular Plant Breeding,2005,3(3):363-368(in Chinese with English abstract).
[16] 徐春波,王 勇,李兴酉,等.含有拟南芥转录因子CBF4基因植物表达载体的构建[J].草业科学,2010,27(6):88-92.XU CH B,WANG Y,LI X Y,et al.Construction of plant expression vector containing Arabidopsistranscription factor CBF4[J]Pratacultural Science,2010,27(6):88-92(in Chinese with English abstract).
[17] 徐春波,王 勇,赵来喜,等.基因枪法获得转CBF4基因蒙农杂种冰草的研究[J].中国草地学报,2013,35(5):24-28.XU CH B,WANG Y,ZHAO L X,et al.Studies on transformation of Agropyron cristatum×A.desertorum cv.Mengnong with CBF4 by particle bombardment[J].Chinese Journal of Grassland,2013,35(5):24-28(in Chinese with English abstract).
[18] 李利斌,刘立锋,王殿峰,等.大白菜CBF4基因的克隆和遗传进化分析[J].山东农业科学,2009(4):1-4.LI L B,LIU L F,WANG D F,et al.Cloning and phylogeny analysis of Chinese cabbage gene CBF4[J].Shandong Agricultural Sciences,2009(4):1-4(in Chinese with English abstract).
[19] DUTT M,MADHAVARAJ J,GROSSER J W,et al.Agrobacterium tumefaciens-mediated genetic transformation and plant regeneration from a complex tetraploid hybrid citrus root stocks[J].Scientia Horticulturae,2010,123(4):454-458.
[20] PUSHYAMI B,BEENA M R,SINBA M K,et al.In vitro regeneration and optimization of conditions for Agrobacterium-mediated transformation in jute,Corchorus capsularis[J].Journal of Plant Biochemistry and Bioteclmology,2011,20(1):39-46.
[21] YEPES I M,MITTAK V,SLIGHTOM J I,et al.Agrobacterium tumefaciens versus biolistic mediated transformation of the Chrysanthemum cv.Polaris and Golden Polaris with nuleocapsid protein genes of three tospovirus speciesm[J].Acta Horticulturae,1999,482:209-218.
[22] 崔新利,陈发棣,陈素梅.地被菊雨花勋章再生和遗传转化体系的建立[J].南京农业大学学报,2009,32(2):40-46.CUI X L,CHEN F L,CHEN S M.Establishment of regeneration and transtormation system of groud-cover Chrysanthemum Yuhuaxunzhang[J].Journal of Nanjing Agricultural University,2009,32(2):40-46(in Chinese with English abstract).
[23] 许志茹,陈智华,姜艳东,等.露地菊离体再生体系建立及BrDFR基因遗传转化[J].园艺学报,2013,40(8):1517-1526.XU ZH R,CHEN ZH H,JIANG Y D,et al.Establishment of the regeneration system and genetic transformation of BrDFRgene in Chrysanthemumcultivar[J].Acta Horticulturae Sinica,2013,40(8):1517-1526(in Chinese with English abstract).
[24] DA SLILVA J A T,SHINOYAMA H,AIDA R,et al.Chrysanthemum biotechnology:Quo vadis?[J].Critical Reviews in Plant Sciences,2013,32(1):21-52.
[25] 郭惠明,李召春,张 晗,等.棉花CBF基因的克隆及其转基因烟草的抗寒性分析[J].作物学报,2011,37(2):286-293.GUO H M,LI ZH CH,ZHANG H,et al.Cloning of cotton CBF gene and its cold tolerance expression in transgenic tobacco[J].2011,37(2):286-293(in Chinese with English abstract).
[2] 陈发棣,房伟民,赵宏波,等.菊花新品种-地被菊系列[J].园艺学报,2005(6):1167-1168.CHEN F L,FANG W M,ZHAO H B,et al.New varieties of Chrysanthemum-Ground Cover Varieties[J].Acta Horticulturae Sinica,2005(6):1167-1168(in Chinese with English abstract).
[3] 张晓娇,史春凤,李春水,等.转AtDREB1A基因地被菊杂交后代优株耐寒性分析[J].园艺学报,2011,38(9):1717-1726.ZHANG X J,SHI CH F,LI CH SH,et al.Cold stress tolerance of the filial generations produced by AtDREB1Atransgenic ground cover Chrysanthemumand a conventional variety[J].Acta Horticulturae Sinica,2011,38(9):1717-1726(in Chinese with English abstract).
[4] MORRAN S,EINI O,PYVOVARENKO T,et al.Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors[J].Plant Biotechnol Journal,2011,9(2):230-249.
[5] 翟俊峰,王法微,王 南,等.月季CBF转录因子基因的克隆及表达分析[J].园艺学报,2012,39(8):1596-1602.ZHAI J F,WANG F W,WANG N,et al.Cloning and expression profiling of the transcription factor CBFgene from rosa hybrida[J].Acta Horticulturae Sinica,2012,39(8):1596-1602(in Chinese with English abstract).
[6] SHAN W,KUANG J F,LU W J,et al.Banana fruit NAC transcription factor MaNACl is a direct target of MaICE1and involved in cold stress through interacting with MaCBF1[J].Plant Cell &Environment,2014.37(9):2116-2127.
[7] CHEN M,WANG Q Y,CHENG X G,et al.GmDREB2,a soybean DRE-Binding transcription factor,conferred drought and high-salt tolerance in transgenic plants[J].Biochemical and Biophysical Research Communications,2007,353(2):299-305.
[8] ZHOU M Q,SHEN C,WU L H,et al.CBF-dependent signaling pathway:a key responder to low temperature stress in plants[J].Critical Reviews in Biotechnology,2011,31(2):186-192.
[9] 刘 杰.甘菊ClCBF家族基因分离及其对拟南芥的转化[D].哈尔滨:东北林业大学,2013.LIU J.Isolation of CBFfamily genes fromChrysanthemum lavandulifolium and transformation studies of ClCBFin Arabidopsis thaliana[D].Harbin:Northeast Forestry University,2013(in Chinese with English abstract).
[10] 肖 政,范崇辉,金万梅.根癌农杆菌介导转录因子CBF1基因对菊花的转化[J].西北农业学报,2009,18(6):262-267.XIAO ZH,FAN CH H,JIN W M.Agrobacterium-mediated transformation of Deranthema morifolium Tzvel.cv.Xiaojinhuang with transcription factor CBF1 gene[J]Acta Agriculturae Boreali-Occidentalis Sinica,2009,18(6):262-267(in Chinese with English abstract).
[11] 向太和,王 琳,蒋 欢,等.发根农杆菌K599对菊花活体转化及其高效再生[J].园艺学报,2011,38(7):1365-1370.XIANG T H,WANG L,JIANG H,et al.Hairy roots induced by Agrobacterium thizogenes K599in Chrysanthemumin vivo and plant regeneration from hairy roots[J].Acta Horticulturae Sinica,2011,38(7):1365-1370(in Chinese with English abstract).
[12] 储 俊,许 娜,张 强,等.农杆菌介导的药百合鳞片遗传转化体系的建立[J].草业学报,2011,20(6):164-169.CHU J,XU N,ZHANG Q,et al.Establishment of an Agrobacterium tumefaciens-mediated genetic transformation system of Lilium speciosum[J].Acta Prataculturae Sinica,2011,20(6):164-169(in Chinese with English abstract).
[13] SUN H J,UCHII S,WATANABE S,et al.A highly efficient transformation protocol for Micro-Tom,a model cultivar for tomato functional genomics[J].Plant Cell Physiol,2006,47(3):426-431.
[14] KNIGHT M R,KNIGHT H.Low-temperature perception leading to gene expression and cold tolerance in higher plants[J].New Phytologist,2012,195(4):737-751.
[15] 霍秀文,米福贵,云锦凤,等.转录因子CBF4诱导型启动子的克隆及功能分析[J].分子植物育种,2005,3(3):363-368.HUO X W,MI F G,YUN J F,et al.Clonging and functional analysis of an induced promoter of transcriptional factor CBF4 from Arabidopsis[J].Molecular Plant Breeding,2005,3(3):363-368(in Chinese with English abstract).
[16] 徐春波,王 勇,李兴酉,等.含有拟南芥转录因子CBF4基因植物表达载体的构建[J].草业科学,2010,27(6):88-92.XU CH B,WANG Y,LI X Y,et al.Construction of plant expression vector containing Arabidopsistranscription factor CBF4[J]Pratacultural Science,2010,27(6):88-92(in Chinese with English abstract).
[17] 徐春波,王 勇,赵来喜,等.基因枪法获得转CBF4基因蒙农杂种冰草的研究[J].中国草地学报,2013,35(5):24-28.XU CH B,WANG Y,ZHAO L X,et al.Studies on transformation of Agropyron cristatum×A.desertorum cv.Mengnong with CBF4 by particle bombardment[J].Chinese Journal of Grassland,2013,35(5):24-28(in Chinese with English abstract).
[18] 李利斌,刘立锋,王殿峰,等.大白菜CBF4基因的克隆和遗传进化分析[J].山东农业科学,2009(4):1-4.LI L B,LIU L F,WANG D F,et al.Cloning and phylogeny analysis of Chinese cabbage gene CBF4[J].Shandong Agricultural Sciences,2009(4):1-4(in Chinese with English abstract).
[19] DUTT M,MADHAVARAJ J,GROSSER J W,et al.Agrobacterium tumefaciens-mediated genetic transformation and plant regeneration from a complex tetraploid hybrid citrus root stocks[J].Scientia Horticulturae,2010,123(4):454-458.
[20] PUSHYAMI B,BEENA M R,SINBA M K,et al.In vitro regeneration and optimization of conditions for Agrobacterium-mediated transformation in jute,Corchorus capsularis[J].Journal of Plant Biochemistry and Bioteclmology,2011,20(1):39-46.
[21] YEPES I M,MITTAK V,SLIGHTOM J I,et al.Agrobacterium tumefaciens versus biolistic mediated transformation of the Chrysanthemum cv.Polaris and Golden Polaris with nuleocapsid protein genes of three tospovirus speciesm[J].Acta Horticulturae,1999,482:209-218.
[22] 崔新利,陈发棣,陈素梅.地被菊雨花勋章再生和遗传转化体系的建立[J].南京农业大学学报,2009,32(2):40-46.CUI X L,CHEN F L,CHEN S M.Establishment of regeneration and transtormation system of groud-cover Chrysanthemum Yuhuaxunzhang[J].Journal of Nanjing Agricultural University,2009,32(2):40-46(in Chinese with English abstract).
[23] 许志茹,陈智华,姜艳东,等.露地菊离体再生体系建立及BrDFR基因遗传转化[J].园艺学报,2013,40(8):1517-1526.XU ZH R,CHEN ZH H,JIANG Y D,et al.Establishment of the regeneration system and genetic transformation of BrDFRgene in Chrysanthemumcultivar[J].Acta Horticulturae Sinica,2013,40(8):1517-1526(in Chinese with English abstract).
[24] DA SLILVA J A T,SHINOYAMA H,AIDA R,et al.Chrysanthemum biotechnology:Quo vadis?[J].Critical Reviews in Plant Sciences,2013,32(1):21-52.
[25] 郭惠明,李召春,张 晗,等.棉花CBF基因的克隆及其转基因烟草的抗寒性分析[J].作物学报,2011,37(2):286-293.GUO H M,LI ZH CH,ZHANG H,et al.Cloning of cotton CBF gene and its cold tolerance expression in transgenic tobacco[J].2011,37(2):286-293(in Chinese with English abstract).