拟南芥GDP-甘露糖焦磷酸化酶基因转化生菜的研究

Transformation of GDP-mannose Pyrophosphorylase Gene from Arabidopsis Thaliana L. into Lactuca Sativa L.

维生素C是植物体内重要的抗氧化物质,同时也是人体所必需的营养物质。为了提高生菜中维生素C的含量,根据GenBank中拟南芥GDP-甘露糖焦磷酸化酶基因(GMP)序列设计特异引物,从拟南芥cDNA中扩增出了GMP基因编码区片段,分别连上CaMV 35S启动子、MYC序列和NOS终止子后将含GMP基因的表达盒插入到植物表达载体pCAMBIA2301中,获得含有GMP基因的植物表达载体p2301-GMP-myc。通过农杆菌介导法转化生菜,获得了64株转基因植株,PCR检测以及荧光定量PCR分析证实外源基因已被成功导入生菜基因组中并表达。采用HPLC-ELSD测定转基因生菜中维生素C的含量。结果表明,大多数转基因生菜中维生素C的含量高于对照植株。转基因生菜中维生素C含量最高的约为对照的2.5倍。该研究证明过量表达GMP基因是提高生菜中维生素C含量的有效方法。

Vitamin C is an important antioxidant in plants and also an indispensable nutriment for human beings.In order to improve the content of vitamin C in Lactuca sativa L.,GDP-mannose pyropho-sphorylase（GMP） gene was cloned with specific primers according to the GenBank sequence from cDNA of Arabidopsis thaliana.CaMV 35S promoter,MYC sequence and NOS terminator were connected to GMP gene respectively to constitute a GMP-containing expression cassette,the latter was transferred into pCAMBIA2301 expression vector resulting in p2301-GMP-myc.L.sativa was transformed with Agrobacterium strain EHA105 containing p2301-GMP-myc through Agrobacterium-mediated leaf-disc transformation,and 64 transgenic plants were obtained.PCR and real-time RT-PCR confirmed the integration and expression of the introduced GMP in transgenic L.sativa plants.The contents of vitamin C in transgenic L.sativa plants were measured by HPLC-ELSD.The results indicated that most of the transgenic plants had higher vitamin C contents than wild type plants,with the highest content of vitamin C about 2.5 times of the wild type.This study demonstrated that over-expressing the GMP gene is an effective method to enhance vitamin C content in L.sativa L.