棉花转基因“喷花法”应用条件优化的研究  

Optimization of Application Condition of “An injection Method” in Cotton Gene Transformation

作　　者：聂新辉 尤春源 雷江荣 杨洋[2] 李波[2] 李晓东[2] 吕军 冯国礼 孔杰[3] 张大伟[3] 

机构地区：[1]新疆石河子农业科技开发研究中心/棉花研究所,新疆石河子832000 [2]新疆农业科学院核技术生物技术研究所,新疆乌鲁木齐830091 [3]新疆农业科学院经济作物研究所,新疆乌鲁木齐830091

出　　处：《作物杂志》2013年第6期31-35,共5页Crops

基　　金：新疆兵团青年创新基金项目(2010JC26);国家转基因重大专项(2011ZX08005-002)

摘　　要：针对目前众多的棉花转基因方法存在转化过程复杂、成本高、效率较低等问题，实验对转基因方法一“喷花法”进行优化，明确了在棉花育种科研中应用条件，达到快速、高效转化外源基因的目的。实验选用了5个陆地棉品种（系）和2个海岛棉品种为转化受体材料，转化了2个外源基因-抗虫（Bt＋CpTI）和增强纤维品质基因（NPTII-GhCAD6），并对转化时等渗液各成分的比例、转化的时间段、温度及湿度条件等因素对转化率的影响进行研究。结果表明，转化效率最高时等渗液各成分的含量：陆地棉为1／2MS＋10％蔗糖＋0．5g／LMES＋0．01mg／L6-BA＋200μl／／L表面活性剂，pH=6菌株浓度OD600=0．5；海岛棉为1／2MS＋15％蔗糖＋0．5g／LMES＋0．01mg／L6-BA＋200μl／L表面活性剂，pH=6菌株浓度OD600=0．5；转化效率最高时转化的时间段为11：00—13130，相应的温度为22～29℃、湿度为70％-85％；转化后的哟代受体材料，首先在田间进行抗生素检测，然后在获得的阳性植株中，转抗虫基因材料用金标BT-CryIAb／Ac试纸条定性检测，转纤维品质基因材料做PCR电泳检测，经检测最终获得了一批转基因阳性植株。最终建立一种应用在农业育种科研中大批量、高效快速、简易实用的转基因方法。Concerning lots of problems about the complex process, high cost and low efficiency in numerous cotton gene transformation methods at present, this experiment was to optimize the application condition of an injection method, and the aims were to clear about the application conditions in the cotton breeding research and achieve rapid and effective transformation methods of exogenous genes. Five upland cotton varieties （ or lines） and two sea island cotton varieties were used as transformation materials in this experiment; two exogenous genes, i. e. , insectresistant （Bt ＋ CpTI） and fiber quality enhancement （ NPTII-GbCAD6 ） gene were transformed, and the factors of conversion efficiency influence on proportion of isotonic solution each component, transformation time, temperature and humidity conditions were studied. The results showed, when the conversion efficiency was the highest, that the contents of isotonic solutions,for upland cotton,were 1/2 MS ＋ 10% Sucrose ＋0.5g/L MES ＋0.01mg/L 6-BA ＋ 200μl/L surfactant,pH = 6 and bacterial strain concentration OD600 = 0.5;for sea island cotton, the contents were 1/2 MS ＋ 15% Sucrose ＋ 0.5g/L MES ＋ 0.01mg/L 6-BA ＋ 200μl/L surfactant, pH = 6 and bacterial strain concentration OD600 =0.5. When conversion efficiency was the highest,the transformation period was 11：00 -13： 30; the corresponding temperature was 22 -29℃ ;the humidity was 70% -85%. After TO generation material were con- verted, which were detected firstly by antibiotics, and then in the obtained positive plants, the transgenic materials of insect-resistant genes were qualitatively detected by Lateral Flow Strip of Bt-CrylAb/Ac ;the transgenic materials of fiber quality enhancement gene were detected by the PCR and electrophoresis. A batch of transgenic positive plants was obtained by this detected method. Finally, a cotton gene transformation method which was mass, efficient, rapid, simple and practical method used in breeding and agricultural scientific research were establ

关 键 词：棉花 转基因 “喷花法” 等渗液 转化条件 

分 类 号：S562[农业科学—作物学]