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1、棉花学报CottonScience2016,28(4):418―424转新型抗草甘膦基因棉花的转化效率及相关分子特征分析11122311121马燕斌袁孙璇袁吴霞袁林朝阳袁刘程毅袁王霞袁张树伟袁王新胜袁张林水袁沈志成袁李燕娥渊1.山西省农业科学院棉花研究所袁山西运城044000曰2.浙江大学农业与生物技术学院袁浙江杭州310029曰3.运城学院生命科学系袁山西运城044000冤摘要院培育抗草甘膦除草剂棉花品种对棉花生产具有重要意义遥利用农杆菌介导法获得了大量抗除草剂草甘膦棉花的再生植株袁对其进行阳性检测遥通过确定PCR检测目标条带大小尧
2、蛋白杂交条带大小分子特征袁对65个独立转化事件在DNA水平尧蛋白水平进行分析和筛选遥结果表明袁经PCR检测再生植株DNA得到的阳性率平均值为81.5%袁经过蛋白检测后阳性率确定为55.4%袁该类阳性植株即为外源基因正常表达的转基因植株遥在此基础上袁结合田间抗性检测袁对其中9个株系进行了不同世代的选择袁经过综合鉴定筛选袁发现2个抗性优良的转基因株系具有极高的利用价值袁其余还需进一步筛选鉴定遥关键词院棉花曰抗草甘膦基因曰转化效率曰阳性率曰抗性表现中图分类号院S562.03文献标志码院A文章编号院1002-7807渊2016冤04-0418
3、-0710.11963/issn.1002-7807.2016040151112231MaYanbin,SunXuan,WuXia,LinChaoyang,LiuChengyi,WangXia,ZhangShuwei,WangXin-1121sheng,ZhangLinshui,ShenZhicheng,LiYan'e(1.044000,;2.310029,;3.044000,)Itisimportanttocultivatetransgeniccottonvarietieswithglyphosate-resistancetoenh
4、ancecottonproductioninChina.Thisresearchaimedtoobtaintransgeniccottonwithanewglyphosate-resistantgenethatwasinitiallyobtainedbyZhejiangUni-versity.Allofthe65independenttransgeniceventsoftheregenerationcottonplantswereobtainedthroughthetissueculturetechnologyusingaglypho
5、sate-resistantscreen.Forthesetransgenicregenerationplants,improvingthetransgenicefficiencyoftheregeneratedplantisveryimportantintransgenicresearch.First,a1.7-kbfragmentofexogenousgeneofall65regenerationplantswasdetectedrespectivelythroughaspecialprimerofG10evo-EPSPSwith
6、PCRamplification.Theresultshowedthattherateofpositivetransgenicplantscouldreachthehighlevelof66.7%―100%,withtheaveragevalueof81.5%.WethenusedthesefragmentcharacterstoverifythepositivesegregatingprogeniesoftransgeniccottonwithPCRamplification.Inaddition,proteinantibodies
7、wereusedinthewestern-blotexperimentforselectingtheresistantplants,inwhichthe46-kDahybridizationbandsweretested.Thestatisticalresultofthetestshowedthatthepositivetransgenicplantsratereached55.4%.BasedonthepositivelinesverifiedbyPCRandwestern-blot,thegenerationsoftransgen
8、iccottonwereselectedbysprayingglyphosateherbi-cidesontothetestfield.Onthisbasis,twotransgeniclineswithexcellen
