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草业学报  2011 

多年生黑麦草P5CS基因的定点突变及其在拟南芥中的转化

, PP. 242-247

Keywords: 多年生黑麦草,定点突变技术,脯氨酸合成酶基因,农杆菌转化

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Abstract:

在前期克隆获得LpP5CS基因的全长cDNA序列基础上,采用PCR扩增技术对多年生黑麦草脯氨酸合成酶基因P5CS的定点突变体系进行了探讨,并运用农杆菌转化技术对突变后的基因进行了功能验证。根据突变位点序列设计一对引物,使用Pfu高保真DNA聚合酶和超级感受态细胞DMT,通过PCR扩增,获得含有所要突变位点的LpP5CSF128A,定向克隆入真核表达载体pCAMBIA1300中,转化拟南芥验证基因功能。结果表明,预期位点上发生了突变,LpP5CS编码的第128位密码子已由苯丙氨酸残基(Phenylalanine,简称Phe或F)变为丙氨酸残基(Alanine,Ala),证明用PCR技术已成功地使LpP5CS基因发生定点突变。转基因拟南芥T1代植株进行PCR和RT-PCR检测,获得4个转基因阳性株系。拟南芥T2代植株经100mmol/LNaCl处理7d后,转基因株系脯氨酸含量分别为4262和5623μg/gFW,显著高于野生型株系的2581μg/gFW。说明转基因株系能积累更多地脯氨酸。

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