多年生黑麦草P5CS基因的定点突变及其在拟南芥中的转化

 在前期克隆获得Lp5CS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犔狆犘５犆犛Ｆ１２８Ａ，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，Lp5CS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Lp5CS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１００ ｍｍｏｌ／Ｌ ＮａＣｌ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。

Site directed mutagenesis of the LpP5CS gene of Lolium perenne and its transformation in Arabidopsis thaliana

Full-length cDNA sequences of the LpP5CS gene,previously obtained using PCR amplification were used as a basis for developing a site-directed mutagenesis system of the Lolium perenne proline synthetase gene LpP5CS.The gene was used in Agrobacterium-mediated transformation technology to verify a functional mutant gene.A pair of primers was designed to the mutation sequence and was used with Pfu high-fidelity DNA polymerase and super-competent cells DMT in PCR amplification.The LpP5CSF128A mutant gene which c...