多年生黑麦草P5CS基因的定点突变及其在拟南芥中的转化  

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

作　　者：曹丽[1,2] 义鸣放[1] 孙振元[2] 韩蕾[2] 巨关升[2] 马欣荣[3] 

机构地区：[1]中国农业大学观赏园艺与园林系,北京100193 [2]中国林业科学研究院林业研究所/国家林业局林木培育重点实验室,北京100091 [3]中国科学院成都生物研究所,四川成都610041

出　　处：《草业学报》2011年第1期242-247,共6页Acta Prataculturae Sinica

基　　金：国家十一五科技支撑计划(2006BAD01A19-2);国家"863"项目(2006AA100109)资助

摘　　要：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/L NaCl处理7d后,转基因株系脯氨酸含量分别为4 262和5 623μg/g FW,显著高于野生型株系的2 581μg/g FW。说明转基因株系能积累更多地脯氨酸。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 contained the desired mutational sites was directly cloned into the eukaryotic expression vector pCAMBIA1300,for transformation into Arabidopsis for validation of gene function.The expected locus mutations had occurred in LpP5CS and changed the encoding codon No.128 from a phenylalanine residue（Phe or F） into an alanine residue（Alanine,Ala）.This was proof that PCR technology had succeeded in site-directed mutagenesis of the LpP5CS gene.Four transgenic lines were obtained from the T1 generation of transgenic Arabidopsis plants by PCR and RT-PCR.The T2 generation of Arabidopsis plants were treated with 100 mmol/L NaCl solution for 7 d and the proline content of T1 and T2 transgenic plants were 4 262 and 5 623 μg/g FW respectively,higher than the 2 581 μg/g FW of wild-type strains.This indicated that transgenic strains can accumulate more proline.

关 键 词：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 

分 类 号：S543.603.53[农业科学—作物学] Q943.2[生物学—植物学]