首页 | 本学科首页   官方微博 | 高级检索  
     

多年生黑麦草P5CS基因的定点突变及其在拟南芥中的转化
引用本文:曹丽,义鸣放,孙振元,韩蕾,巨关升,马欣荣. 多年生黑麦草P5CS基因的定点突变及其在拟南芥中的转化[J]. 草业学报, 2011, 20(1): 242. DOI: 10.11686/cyxb20110133
作者姓名:曹丽  义鸣放  孙振元  韩蕾  巨关升  马欣荣
作者单位:曹丽,CAO Li(中国农业大学观赏园艺与园林系,北京,100193;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);义鸣放,YI Ming-fang(中国农业大学观赏园艺与园林系,北京,100193);孙振元,韩蕾,巨关升,SUN Zhen-yuan,HAN Lei,JU Guan-sheng(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);马欣荣,MA Xin-rong(中国科学院成都生物研究所,四川,成都,610041)
基金项目:国家十一五科技支撑计划,国家"863"项目
摘    要: 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。

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
收稿时间:1900-01-01;

Site directed mutagenesis of the LpP5CS gene of Lolium perenne and its transformation in Arabidopsis thaliana
CAO Li,YI Ming-fang,SUN Zhen-yuan,HAN Lei,JU Guan-sheng,MA Xin-rong. Site directed mutagenesis of the LpP5CS gene of Lolium perenne and its transformation in Arabidopsis thaliana[J]. Acta Prataculturae Sinica, 2011, 20(1): 242. DOI: 10.11686/cyxb20110133
Authors:CAO Li  YI Ming-fang  SUN Zhen-yuan  HAN Lei  JU Guan-sheng  MA Xin-rong
Affiliation:CAO Li 1,2,YI Ming-fang1,SUN Zhen-yuan2,HAN Lei 2,JU Guan-sheng2,MA Xin-rong3(1.Department of Ornamental Horticulture and Landscape Architecture,China Agriculture University,Beijing 100193,China,2.Research Institute of Forestry,Chinese Academy of ForestryKey Laboratory of Tree Breeding and Cultivation,State Forestry Administration,Beijing 100091,3.Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China)
Abstract: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...
Keywords:Lolium perenne  site-directed mutagenesis  proline synthase genes  Agrobacterium-mediated transformation technology  
本文献已被 CNKI 万方数据 等数据库收录!
点击此处可从《草业学报》浏览原始摘要信息
点击此处可从《草业学报》下载全文
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号