棕色棉茎尖基因枪遗传转化
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摘要
为了建立高效的棉花茎尖遗传转化体系,以棕色棉品种新彩棉5号为材料,采用茎尖培养和基因枪遗传转化方法,通过对凝固剂种类、活性炭含量、卡那霉素浓度、渗透处理时间、轰击压力、轰击距离等因素的优化,建立了棕色棉新彩棉5号茎尖基因枪遗传转化技术体系。结果表明,不同凝固剂种类、活性炭含量、卡那霉素浓度、渗透处理时间、轰击压力和轰击距离对棕色棉茎尖转化率有明显影响。最佳的遗传转化体系为:前渗处理4 h,轰击压力7.586 MPa(1100 psi),轰击距离9 cm,轰击后恢复培养4 d,然后在MSB+8 g·L~(-1)琼脂+125mg·L~(-1)卡那霉素筛选培养基中筛选25 d。获得抗性芽后,在添加1 g·L~(-1)活性炭的生根培养基中诱导抗性芽生根,并获得抗性再生植株。通过PCR和q PCR检测,5200个茎尖共获得16株阳性转基因植株,转化率为0.31%。该技术体系的建立为棕色棉的遗传转化奠定了基础,为彩色棉育种提供了新的材料。
To genetically transform cotton shoot tips, we established a biolistic genetic transformation system though the plant shoot tip culture technique and the gene gun-mediated transformation method, using the shoot tips of brown cotton Xincaimian No.5, a Gossypium hirsutum L. variety cultured in Xinjiang as materials. Several critical factors of the system were assessed,including the type of coagulant, the activated charcoal concentration, the kanamycin concentration, the penetration processing time, and the bombardment pressure and distance. Results showed that each factor clearly influenced the transformation rate of brown cotton shoot tips. An optimal biolistic genetic transformation technology system was determined:(1) shoot tips were cultured for 4 h before being bombarded in the osmotic medium;(2) the shoot tips were bombarded with helium pressure of7.586 MPa(1100 psi), and a bombardment distance of 9 cm;(3) after culturing for 4 d in the recovery medium, shoot tips were moved to MSB screening medium containing 8 g·L~(-1)agar powder and 125 mg·L~(-1)kanamycin and then screened for 25 d to obtain the resistant germs;(4) then, 1 g·L~(-1)activated charcoal was added to the induction medium to induce rooting of the resistant germs to obtain resistant regenerating plants. After PCR and q PCR detections, 16 plants positive for the transgene were obtained from 5200 shoot tips, with a transformation rate of 0.31%. The establishment of this technology system lays a foundation for the genetic transformation of brown cotton and provides new materials for naturally-colored cotton breeding.
To genetically transform cotton shoot tips, we established a biolistic genetic transformation system though the plant shoot tip culture technique and the gene gun-mediated transformation method, using the shoot tips of brown cotton Xincaimian No.5, a Gossypium hirsutum L. variety cultured in Xinjiang as materials. Several critical factors of the system were assessed,including the type of coagulant, the activated charcoal concentration, the kanamycin concentration, the penetration processing time, and the bombardment pressure and distance. Results showed that each factor clearly influenced the transformation rate of brown cotton shoot tips. An optimal biolistic genetic transformation technology system was determined:(1) shoot tips were cultured for 4 h before being bombarded in the osmotic medium;(2) the shoot tips were bombarded with helium pressure of7.586 MPa(1100 psi), and a bombardment distance of 9 cm;(3) after culturing for 4 d in the recovery medium, shoot tips were moved to MSB screening medium containing 8 g·L~(-1)agar powder and 125 mg·L~(-1)kanamycin and then screened for 25 d to obtain the resistant germs;(4) then, 1 g·L~(-1)activated charcoal was added to the induction medium to induce rooting of the resistant germs to obtain resistant regenerating plants. After PCR and q PCR detections, 16 plants positive for the transgene were obtained from 5200 shoot tips, with a transformation rate of 0.31%. The establishment of this technology system lays a foundation for the genetic transformation of brown cotton and provides new materials for naturally-colored cotton breeding.
引文
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[14]刘传亮,于娅,王玉芬,等.棉花基因枪茎尖转化研究[J].棉花学报,2003,15(6):361-366.Liu Chuanliang,Yu Ya,Wang Yufen,et al.Study on transformation of meristem via gene-gun bombardment[J].Cotton Science,2003,15(6):361-366.
[15]耿立召.棉花基因枪转化体系的建立[D].北京:中国农业科学院,2004.Geng Lizhao.The establishment of transformation system of cotton(Gossypium hirsutum L.)via particle bombardment[D].Beijing:Chinese Academy of Agricultural Sciences,2004.
[16]马玲玲,魏延宏,何兰兰,等.基因枪介导的海岛棉(Gossypium barbadense L.)茎尖遗传转化体系的建立[J].棉花学报,2014,26(3):213-220.Ma Lingling,Wei Yanhong,He Lanlan,et al.The establishment of biolistics transformation system of the apical meristem of Gossypium barbadense L.[J].Cotton Science,2014,26(3):213-220.
[17]萨姆布鲁克J,拉塞尔D W.分子克隆试验指南[M].3版.金冬雁,黎孟枫,译.北京:科学出版社,1999:19-22.Sambrook J,Russell D W.Molecular cloning:a laboratory manual[M].3rd ed.Translated by Jin Dongyan,Li Mengfeng.Beijing:Science Press,1999:19-22.
[18]赵福永.棉花茎尖培养及高效植株再生体系的建立[J].长江大学学报,2008,5(4):74-77.Zhao Fuyong.The establishment of efficient plant regeneration system and cotton shoot apical culture[J].Journal of Yangtze University,2008,5(4):74-77.
[19]李雪.基因枪介导的棉花遗传转化及后代遗传分析[D].保定:河北农业大学,2007.Li Xue.The genetic transformation of cotton via particle bombardment and genetic analysis of generation[D].Baoding:Agricultural University of Hebei,2007.
[20]Liu J F,Wang X F,Li Q L,et al.Biolistic transformation of cotton(Gossypium hirsutum L.)with the phy A gene from Aspergillus ficuum[J].Plant Cell Tissue and Organ Culture,2011,106:207-214.
[21]耿立召,李付广,刘传亮,等.影响基因枪轰击转化棉花胚性愈伤的因素初探[J].棉花学报,2004,16(6):352-356.Geng Lizhao,Li Fuguang,Liu Chuanliang,et al.Elementary exploration on factors of influencing transformation via particle bombardment embryonic calluses of cotton[J].Cotton Science,2004,16(6):352-356.●
[2]赵兴华,渠云芳,黄晋玲.彩色棉育种研究现状与展望[J].现代农业科技,2011(5):84-85.Zhao Xinghua,Qu Yunfang,Huang Jinling.Present situation and prospect of colored cotton breeding[J].Modern Agricultural Science and Technology,2011(5):84-85.
[3]朱进忠,徐亚平.棉纺织品的生态设计[J].棉纺织技术,2014,42(1):74-77.Zhu Jinzhong,Xu Yaping.Cotton textiles ecological design[J].Cotton Textile Technology,2014,42(1):74-77.
[4]尤春源,聂新辉,吕军,等.新疆彩色棉育种进展现状及存在的问题[J].中国棉花,2011,38(2):6-7.You Chunyuan,Nie Xinhui,LüJun,et al.Progress and problems of naturally-colored cotton breeding in Xinjiang[J].China Cotton,2011,38(2):6-7.
[5]路正营,李世云,韩永亮,等.转基因技术在棉花遗传改良中的应用[J].河北农业科学,2011,15(2):92-95.Lu Zhengying,Li Shiyun,Han Yongliang,et al.Application of transgenic technology in genetic improvement of cotton[J].Journal of Hebei Agricultural Sciences,2011,15(2):92-95.
[6]冯克云.基因工程技术在彩色棉育种中的应用概况[J].甘肃农业科技,2008(2):30-32.Feng Keyun.Application of gene engineering technology in colored cotton to breeding[J].Gansu Agricultural Science and Technology,2008(2):30-32.
[7]Zhang Kang,Liu Jinxing,Zhang Yi,et al.Biolistic genetic transformation of a wide range of Chinese elite wheat(Triticum aestivum L.)varieties[J].Journal of Genetics and Genomics,2015,42(1):39-42.
[8]Nguyen T X,Sticklen M.Barley HVA1 gene confers drought and salt tolerance in transgenic maize(Zea mays L.)[J/OL].Advances in Crop Science and Technology,2013,1(1):1000105[2015-09-28].http://dx.doi.org/10.4172/acst.1000105.
[9]Wang Ying,Cao Liming,Zhang Yuexiong,et al.Map-based cloning and characterization of BPH29,a B3 domain-containing recessive gene conferring brown planthopper resistance in rice[J].Journal of Experimental Botany,2015,66(19):6035-6045.
[10]Trolinder N L.The role of biotechnology in cotton improve ment[C]//National Cotton Council of USA.Proceedings of beltwide cotton production research conferences.Memphis,TN,USA:National Cotton Council,1989:636-637.
[11]Dennis E,Brian J M.Transformation of elite cotton cultivar via particle bombardment of meristems[J].Biotechnology,1993,11(5):596-598.
[12]朱卫民,吴敬音,佘建明,等.棉花茎尖分生组织在微粒轰击法基因转化中的应用[J].江苏农业学报,1998,14(2):74-79.Zhu Weiming,Wu Jingyin,She Jianming,et al.Application of the shoot apical meristem of cotton in gene transformation by particle bombardment[J].Jiangsu Journal of Agricultural Sciences,1998,14(2):74-79.
[13]于娅,刘传亮,马峙英,等.适于基因枪转化的棉花茎尖培养及筛选体系初探[J].棉花学报,2003,15(5):274-278.Yu Ya,Liu Chuanliang,Ma Zhiying,et al.The culture of cotton apical meristem and its application in particle bombardment transformation[J].Cotton Science,2003,15(5):274-278.
[14]刘传亮,于娅,王玉芬,等.棉花基因枪茎尖转化研究[J].棉花学报,2003,15(6):361-366.Liu Chuanliang,Yu Ya,Wang Yufen,et al.Study on transformation of meristem via gene-gun bombardment[J].Cotton Science,2003,15(6):361-366.
[15]耿立召.棉花基因枪转化体系的建立[D].北京:中国农业科学院,2004.Geng Lizhao.The establishment of transformation system of cotton(Gossypium hirsutum L.)via particle bombardment[D].Beijing:Chinese Academy of Agricultural Sciences,2004.
[16]马玲玲,魏延宏,何兰兰,等.基因枪介导的海岛棉(Gossypium barbadense L.)茎尖遗传转化体系的建立[J].棉花学报,2014,26(3):213-220.Ma Lingling,Wei Yanhong,He Lanlan,et al.The establishment of biolistics transformation system of the apical meristem of Gossypium barbadense L.[J].Cotton Science,2014,26(3):213-220.
[17]萨姆布鲁克J,拉塞尔D W.分子克隆试验指南[M].3版.金冬雁,黎孟枫,译.北京:科学出版社,1999:19-22.Sambrook J,Russell D W.Molecular cloning:a laboratory manual[M].3rd ed.Translated by Jin Dongyan,Li Mengfeng.Beijing:Science Press,1999:19-22.
[18]赵福永.棉花茎尖培养及高效植株再生体系的建立[J].长江大学学报,2008,5(4):74-77.Zhao Fuyong.The establishment of efficient plant regeneration system and cotton shoot apical culture[J].Journal of Yangtze University,2008,5(4):74-77.
[19]李雪.基因枪介导的棉花遗传转化及后代遗传分析[D].保定:河北农业大学,2007.Li Xue.The genetic transformation of cotton via particle bombardment and genetic analysis of generation[D].Baoding:Agricultural University of Hebei,2007.
[20]Liu J F,Wang X F,Li Q L,et al.Biolistic transformation of cotton(Gossypium hirsutum L.)with the phy A gene from Aspergillus ficuum[J].Plant Cell Tissue and Organ Culture,2011,106:207-214.
[21]耿立召,李付广,刘传亮,等.影响基因枪轰击转化棉花胚性愈伤的因素初探[J].棉花学报,2004,16(6):352-356.Geng Lizhao,Li Fuguang,Liu Chuanliang,et al.Elementary exploration on factors of influencing transformation via particle bombardment embryonic calluses of cotton[J].Cotton Science,2004,16(6):352-356.●