拟南芥GDPMPPASE和GALPPASE基因转化生菜研究
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摘要
维生素C(L-ascorbic acid,AsA)是维持人体正常生长发育必需的一类微量营养物质,但是人类自身却不能合成以及储存AsA,必须从外界获取。而植物性食物是人体维生素C的重要来源,L-半乳糖途径在植物AsA的生物合成中占有主导地位。GDP-甘露糖焦磷酸化酶(GDPMPPase)和L-半乳糖-1-磷酸化酶(GalPPase)便是L-半乳糖合成途径中两个重要的关键酶, GDPMPPase催化D-甘露糖-1-P生成GDP-D-甘露糖,而GalPPase催化GDP-L-半乳糖-1- P合成L-Gal。为了提高生菜中AsA的含量,根据GenBank中拟南芥GDP-甘露糖焦磷酸化酶基因(gmp)和L-半乳糖-1-磷酸化酶基因(galp)序列设计特异引物,从拟南芥cDNA中扩增出了gmp和galp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/galp基因的表达盒插入到植物表达载体pCAMBIA2301中,获得含有gmp/galp基因的植物表达载体p2301-gmp-myc和p2301-galp-myc。通过农杆菌介导法转化生菜,分别获得了64株转gmp基因植株和56株转galp基因植株,PCR检测以及Southern Blot杂交和荧光定量PCR分析证实外源基因已被成功导入生菜基因组中并转录。采用高效液相色谱法(HPLC-ELSD)测定转基因生菜中AsA的含量,结果表明,大多数转基因生菜中AsA的含量高于对照植株。AsA含量的HPLC分析结果显示:各gmp转基因株系中AsA含量最高达到对照的2.5倍;各galp转基因株系中AsA含量最高达到对照的1.9倍。该研究证明过量表达gmp基因和galp基因是提高生菜中维生素C含量的有效方法。
Vitamin C (L-ascorbic acid, AsA) is an essential micronutrient for human life. However, AsA can not be synthesized and stored in human bodies. AsA is obtained mainly from plants. In plants AsA is synthesized with D-mannose and L-galactose as key intermediates. GDP-D-mannose pyrophosphorylase (GDPMPPASE, EC2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant AsA biosynthesis. L-Galactose-1-phosphatase catalyzes the synthesis of L-Galactose. In order to improve the content of AsA in Lactuca sativa L., GDP-mannose pyrophosphorylase gene (gmp) and L-Galactose-1-phosphatase gene(galp)were 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 /galp gene respectively to constitute a gmp -containing expression cassette and a galp -containing expression cassette, the latter cassettes were transferred into pCAMBIA2301 expression vector respectively resulting in p2301-gmp-myc and p2301-galp-myc. L. sativa was transformed with Agrobacterium strain EHA105 containing p2301-gmp-myc or p2301-galp-myc through Agrobacterium-mediated leaf-disc transformation, and 64 gmp transgenic plants and 56 galp transgenic plants were obtained. PCR, Southern Blot and Real-Time PCR confirmed the integration and expression of the introduced gmp /galp in transgenic L. sativa plants. The contents of AsA in transgenic L. sativa plants were measured by HPLC–ELSD. The results indicated that most of the transgenic plants had higher AsA contents than those of wild type plants, with the highest content of AsA being about 2.5 times(gmp) and 1.9 times(galp) of the wild type. This study demonstrates that over-expressing the gmp /galp gene is an effective method to enhance AsA content in L. sativa L.
Vitamin C (L-ascorbic acid, AsA) is an essential micronutrient for human life. However, AsA can not be synthesized and stored in human bodies. AsA is obtained mainly from plants. In plants AsA is synthesized with D-mannose and L-galactose as key intermediates. GDP-D-mannose pyrophosphorylase (GDPMPPASE, EC2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant AsA biosynthesis. L-Galactose-1-phosphatase catalyzes the synthesis of L-Galactose. In order to improve the content of AsA in Lactuca sativa L., GDP-mannose pyrophosphorylase gene (gmp) and L-Galactose-1-phosphatase gene(galp)were 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 /galp gene respectively to constitute a gmp -containing expression cassette and a galp -containing expression cassette, the latter cassettes were transferred into pCAMBIA2301 expression vector respectively resulting in p2301-gmp-myc and p2301-galp-myc. L. sativa was transformed with Agrobacterium strain EHA105 containing p2301-gmp-myc or p2301-galp-myc through Agrobacterium-mediated leaf-disc transformation, and 64 gmp transgenic plants and 56 galp transgenic plants were obtained. PCR, Southern Blot and Real-Time PCR confirmed the integration and expression of the introduced gmp /galp in transgenic L. sativa plants. The contents of AsA in transgenic L. sativa plants were measured by HPLC–ELSD. The results indicated that most of the transgenic plants had higher AsA contents than those of wild type plants, with the highest content of AsA being about 2.5 times(gmp) and 1.9 times(galp) of the wild type. This study demonstrates that over-expressing the gmp /galp gene is an effective method to enhance AsA content in L. sativa L.
引文
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