玉米耐热DNA聚合酶基因表达载体构建与花粉介导转化.PDF

玉 米 科 学2016，24(1)：43～49 JournalofMaizeSciences 文章编号：1005-0906(2016)01-0043-07 DOI:10.13597/ki.maize.science 玉米耐热DNA聚合酶基因表达载体 构建与花粉介导转化 易文斌，庄丽莎，张 海，郭泽鑫，林伟锋，赵海婵，张雅君，梁雪莲 (仲恺农业工程学院生命科学院，广州 510225) 摘 要：实验构建热稳定性聚合酶的基因表达双元载体，利用花粉介导法将热稳定性DNA聚合酶基因导入玉 米幼胚，获得耐热转基因玉米株系。用热稳定性 DNA聚合酶基因构建表达载体；然后利用花粉介导法进行转化， 花粉收集自仲恺自交系玉米甜1号和自交系糯2号，将花粉与构建好的基因表达载体混合，利用花粉介导法进行转 化；将收集到的T1代种子用PCR凝胶电泳技术和Southern杂交技术进行检测，选出阳性的转化株；在T2代经过田间 耐热压力试验结合测定的玉米生理指标，筛选出9个具有良好耐热性玉米株系。 关键词：玉米；热稳定性；DNA聚合酶；双元载体；花粉介导转化法 中图分类号：S513.034 文献标识码：A ConstructionofPlantExpressionVectorwithTaqDNAPolymerase GeneandGainHeat-tolerantMaizeLines YIWen-bin,ZHUANGLi-sha,ZHANGHai,GUOZe-xin,LINWei-feng, ZHAOHai-chan,ZHANGYa-jun,LIANGXue-lian (CollegeofLifeScience,ZhongkaiUniversityofAgricultureandEngineering,Guangzhou510225,China) Abstract:TaqDNApolymerasegenewasusedtodevelopnewheat-tolerantmaizelines,whichwillpotentially addtotheeconomicvalueandaidincontrollingcroplossesduetoheatstress.First,anexpressionvectorwithTaq DNApolymerasegenewasestablished.Then,accordingtopollen-mediatedtransformation,pollenfromthemaize ZhongKaiinbredlines,ZhongsweetNo.1andZhongglutinousNo.2wascollected,whichwasmixedwiththeestab⁃ lishedexpressionvectorandwasappliedtothemaizesilksfortransformation.Thirdly,harvestedT1generation seedswereconfirmedtobepositivetransformantsbyPCRandSouthernhybridization.Finally,afterscreeningthe T2 generation,byexposuretoheatstressconditionsinthefieldcombinedwithphysiologicalindexes,ninetrans⁃ formedheat-tolerantmaizelineswereobtained. Keywords:Maize;TaqDNApolymerase;Binaryvector;Pollen-mediatedtransformation