农杆菌介导大豆子叶节影响因素的研究
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摘要
为提高大豆遗传转化效率,采用农杆菌介导遗传转化的方法,以大豆子叶节为外植体,研究共培养阶段浸染液浓度及外界培养条件(共培养温度和天数)和培养基添加物对转化效率和芽诱导率的影响。结果显示,浸染液OD600=0.5的诱导率最高,共培养温度为24℃,共培养天数为10 d具有较高转化效率,共培养基中加入一定浓度的单壁碳纳米管(Single-wall carbon nanotube,NM)对诱导卡那霉素抗性不定芽有促进作用。PCR检测表明PHR1基因已整合到T1代大豆基因组中,初步证明可在大豆基因组中稳定遗传。
It was to improve the efficiency of soybean genetic transformation, we used the soybean cotyledon node as explants via Agrobactium-mediated transformation method, and investigated the effect of bacterial concentration, co-culture temperature and time on the transformation efficiency and the rate of bud induction. The results showed that under the OD600 = 0.5 of infection solution concentration, and induction rate was the highest. The conversion efficiency was higher under the environment of co-culture temperature being 24℃ for 10 days. The single-wall carbon nanotubes added to culture medium improved the induction of the adventitious bud with kanamycin-resistance. Moreover, the PCR results indicated that PHR1 gene was integrated into T1 genome generation, preliminarily proving that the heredity of gene in the soybean genome was stable.
It was to improve the efficiency of soybean genetic transformation, we used the soybean cotyledon node as explants via Agrobactium-mediated transformation method, and investigated the effect of bacterial concentration, co-culture temperature and time on the transformation efficiency and the rate of bud induction. The results showed that under the OD600 = 0.5 of infection solution concentration, and induction rate was the highest. The conversion efficiency was higher under the environment of co-culture temperature being 24℃ for 10 days. The single-wall carbon nanotubes added to culture medium improved the induction of the adventitious bud with kanamycin-resistance. Moreover, the PCR results indicated that PHR1 gene was integrated into T1 genome generation, preliminarily proving that the heredity of gene in the soybean genome was stable.
引文
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[17]陆长梅,张超英,温俊强,等.纳米材料促进大豆萌发,生长的影响及其机理研究[J].大豆科学,2002,21:168-172.
[18]Ma LL,Liu C,Qu CX,et al.Rubisco activase m RNA expression in spinach:Modulation by nanoanatase treatment[J].Biological Trace Element Research,2008,122(2):168-178.
[19]Khodakovskaya M,Dervishi E,Mahmood M,et al.Carbon nanotubes are able to penetrate plant seed coat and dramatically affect seed germination and plant growth[J].American Chemical Society Nano,2009,10:3221-3227.
[20]王晓红.单壁碳纳米管对大豆子叶节外植体分化与再生影响的研究[D].北京:中国农业科学院,2013.
[21]杨致荣,王兴春,李西明.高等植物转录因子的研究进展[J].遗传,2004,26(3):403-408.
[22]张彩英,李喜焕,常文锁,等.大豆Gm PHR1基因及其编码的蛋白和应用:中国,ZL 201010528355.X[P].2012-09-05.
[2]任海祥,南海洋,曹东,等.大豆转基因技术研究进展[J].东北农业大学学报,2012,43(7):6-12.
[3]Somers DA,Samac DA,Olhoft PM.Recent advances in legume transformation[J].Plant Physiology,2003,131:892-899.
[4]刘圣君,黄健秋,卫志明.影响农杆菌介导的大豆子叶节遗传转化的因素[J].分子细胞生物学报,2007,40(5):286-292.
[5]刘翠,李喜焕,常文锁,等.农杆菌介导大豆不同外植体遗传转化研究]J].华北农学报,2012,27(3):35-40.
[6]Bertolla F,Kay E,Simonet P.Potential dissemination of antibiotic restistace genes from transgenic plants to microorganisms[J].Infect Control Hosp Epidemiol,2000,21(6):390-393.
[7]Losey JE,Rayor LS,Carter ME.Transgenic pollen harms monarch larvae[J].Nature,1999,399:214-215.
[8]祁永斌,刘庆龙,陆艳婷,等.转基因植物中删除选择标记基因的研究进展[J].浙江农业学报,2014,26(5):1387-1393.
[9]周思军,李希臣,刘昭军,等.大豆农杆菌介导转化系统的优化研究[J].大豆科学,2001,32(4):313-319.
[10]武小霞,李静,刘伟婷,等.大豆农杆菌子叶节转化菌株适宜生长时期及浸染浓度的研究[J].东北农业大学学报,2010,41(1):1-6.
[11]刘海坤卫志明.利用农杆菌介导转化大豆成熟种子胚尖获得转基因植株[J[.植物生理与分子生物学学报,2004,30(6):631-636.
[12]段莹莹,赵琳,陈李淼,等.农杆菌介导的大豆子叶节和下胚轴转化方法的比较及优化[J].大豆科学,2010,29(4):590-593.
[13]Meurer CA,Dinkins RD,Collins GB.Factors affecting soybean cotyledonary node transformation[J].Plant Cell Rap,1988,18:180-186
[14]应珊,何晓薇,王秀荣,寿惠霞.影响农杆菌介导的大豆转化效率的因素研究[J].分子植物育种,2008,6(1):32-40.
[15]Masciangioli T,Zhang WX.Environmental technologies at the nanoscale[J].Environ Sci Technol,2003,37:102a-108a.
[16]吕继涛,张淑贞.人工纳米材料与植物的相互作用:植物毒性、吸收与传输[J].化学进展,2013,25(1):156-163.
[17]陆长梅,张超英,温俊强,等.纳米材料促进大豆萌发,生长的影响及其机理研究[J].大豆科学,2002,21:168-172.
[18]Ma LL,Liu C,Qu CX,et al.Rubisco activase m RNA expression in spinach:Modulation by nanoanatase treatment[J].Biological Trace Element Research,2008,122(2):168-178.
[19]Khodakovskaya M,Dervishi E,Mahmood M,et al.Carbon nanotubes are able to penetrate plant seed coat and dramatically affect seed germination and plant growth[J].American Chemical Society Nano,2009,10:3221-3227.
[20]王晓红.单壁碳纳米管对大豆子叶节外植体分化与再生影响的研究[D].北京:中国农业科学院,2013.
[21]杨致荣,王兴春,李西明.高等植物转录因子的研究进展[J].遗传,2004,26(3):403-408.
[22]张彩英,李喜焕,常文锁,等.大豆Gm PHR1基因及其编码的蛋白和应用:中国,ZL 201010528355.X[P].2012-09-05.