提高花生α-维生素E含量的载体构建及其转化研究
摘要
花生(Arachis hypogaea L.)是世界上四大油料作物之一,在世界范围内的热带和亚热带地区都有种植。中国是世界上最大的花生生产国和消费国。维生素E是一种脂溶性的抗氧化剂,在人体、动物以及植物体内都有重要的生理作用。植物油是人类摄取维生素E的主要途径。植物包括花生在内具有高活性α-维生素E的含量非常低,为了提高花生中维生素E的含量,我们试图通过基因工程的办法改良现有的花生品种。本文报道维生素合成途径中的重要基因尿黑酸合成酶(VE_2)基因、2-甲基6-植基-苯醌甲基转移酶(VE_3)基因和γ-生育酚甲基转移酶(VE_4)基因的克隆,种子特异表达启动子引导的植物表达载体构建,以及这些载体对花生进行遗传转化的研究结果。
1、为了使维生素E相关的基因在花生胚中特异表达,通过PCR的方法在同源性分别为99.8%和100%的T载体中分离得到两个种子特异表达启动子。他们分别是大豆种子储藏蛋白启动子(7S)和油菜种子储藏蛋白启动子(2S)。这两个启动子的长度分别为795bp和1058bp。
2、提取拟南芥(Arabidopsis thaliana)叶子的总RNA,并根据GenBank所发表的VE_2、VE_3和VE_4序列设计引物,通过RT-PCR的方法分离得到这3个基因,他们和GenBank上所发表的序列同源性分别为98%、100%和100%。把这三个基因分别构建到中间表达载体pSPROK中,并且在VE2基因的上游插入7S,在VE3和VE4基因上游分别插入2S,来调控这3个基因在种仁特异表达。然后从这3个载体中酶切出分别带有启动子、基因和终止子的完整表达单元,构建到植物表达载体pCAMBIA1300中,所构建成的植物表达载体分别命名为pCAMBIA1300VE2·7S、pCAMBIA1300VE3·2S和pCAMBIA1300VE4·2S。测序分析显示3个基因的完整表达单元成功构建在植物表达载体pCAMBIA1300中。
3.为了更高效地改良花生中维生素E的合成效率,在单价载体的基础上构建了一个双价植物表达载体pCAMBIA1300 VE2·VE3,它含有VE_2和VE_3各自的启动子和终止子表达单元。为了同时改良花生中维生素E含量和油酸含量,亦构建得到双价植物表达载体pCAMBIA1300AT,它含有VE_4的完整表达单元和FAD2的反义RNA表达单元。
4、将pCAMBIA1300VE4·2S、pCAMBIA1300AT、pCAMBIA13002a等3个植物表达载体通过根癌农杆菌介导对花生进行遗传转化,提取抗性苗DNA进行PCR反应显示,他们的表达单元都已经插入到花生基因组中,各得到转基因苗3株、3株和6株,转化率分别为:2.00%、2.00%和3.85%。由于这些基因都是由种子特异表达启动子所引导,应待转基因苗种子收获后再作进一步检测。
综上所述,本研究构建了5个植物种子特异表达载体用来提高花生得维生素E含量,并将pCAMBIA1300VE4·2S、pCAMBIA1300AT、pCAMBIA13002a等3个载体对花生进行了转化,得到了转基因植株,为改良花生维生素E和油酸含量奠定了基础。
Peanut(Arachis hypogaea L.)is the world's fourth largest oilseed crop grown mainly in subtropical and tropical regions.China is the largest country of peanut production and consumption in the world.Vitamin E is an important class of lipid-soluble compounds with antioxidant activities that plays a very important role in plant,animal and human being.Plant oil is the main resource of the vitamin E in human nutritious.The content of activatedα-tocopherol is very low in plant,including peanut.To improve the rate of vitamin E component in peanut,we have tried to improve peanut vitamin E content by employing gene engineering.We reported here the cloning of homogentisic acid prenyltransferase(VE_2),2-methyl-6-phytylbenzoquinol(VE_3)andα-tocopherol methyltransferase(VE_4)genes,embryo-specific expression vectors construction and their transformation.
To make vitamin E genes express specifically in peanut embryo,two promoters already cloned in T-vectors were isolated with 99.8%and 100%identity to the sequences interested on GenBank,respectively.They are 7S seed storage protein gene promoter of soybean and 2S seed storage protein gene promoter of Brassica napus.The promoters region were 795bps and 1058bps in length.
Extracting total RNA from leaves of Arabidopsis thaliana and designing the primers according to the published sequence of VE_2,VE_3 and VE_4 genes on GenBank, we obtained those three genes,with 97%,100%and 100%identity to the conterpart sequences on GenBank,respectively,by RT-PCR.Those three genes were first constructed on intermediate expressing vector pSPROK with 7S promoter upstream to VE2 gene and 2S promoter upstream to VE3 and VE4 genes,respectively.Then the intact expression units with genes of interest flanked by specific promoters and nos terminals were cut and transferred to plant expressing vector pCAMBIA1300.Three plant expression vectors,named pCAMBIA1300VE2.7S,pCAMBIA1300VE3.2S and pCAMBIA1300VE4.2S,were obtained afterwards.Sequencing results showed that all the three genes were correctly inserted in plant expressing vector pCAMBIA1300.
To improve the tocopherol biosynthetic pathway and to increase the total amount of vitamine E,a plant bivalent expression vector,pCAMBIA1300 VE2.VE3,was constructed.It contains VE_2 and VE_3 genes,each has their own promoter and nos terminal.To improve both tocopherol and the rate of oleic acid component in peanut, another plant bivalent expression vector pCAMBIA1300AT was constructed.It has intact ultra-expression units of VE_4 and complete anti-RNA expression unit of FAD2.
Three vectors,pCAMBIA1300VE4.2S,pCAMBIA1300AT and pCAMBIA13002a, have been used to transform peanut variety Minghua 6,mediated by agrobacterium C58. Through many resistant transgenic seedling were obtained,only three,three and six seedlings with respective to genes of VE2,anti-RNA of FAD2 and VE4,and anti-FAD2 were shown to carry the genes of interest in the cells,after subjected to PCR assay,with transformation rate of 2.0%,2.0%and 3.85%.Further evaluation must be done after seed harvest,since those genes are all directed by seed specifically expressed prmoters.
In a word,we obtained five plant expressing vectors involved in increasing vatamine E contant.Plant expressing vectors pCAMBIA1300VE4-2S, pCAMBIA1300AT and pCAMBIA13002a were being used to transform peanut mediated by Agrobacterium and some transfomant seedlings were got,which provides basis in improving vitamin E and fatty acid composition in peanuts.
1、为了使维生素E相关的基因在花生胚中特异表达,通过PCR的方法在同源性分别为99.8%和100%的T载体中分离得到两个种子特异表达启动子。他们分别是大豆种子储藏蛋白启动子(7S)和油菜种子储藏蛋白启动子(2S)。这两个启动子的长度分别为795bp和1058bp。
2、提取拟南芥(Arabidopsis thaliana)叶子的总RNA,并根据GenBank所发表的VE_2、VE_3和VE_4序列设计引物,通过RT-PCR的方法分离得到这3个基因,他们和GenBank上所发表的序列同源性分别为98%、100%和100%。把这三个基因分别构建到中间表达载体pSPROK中,并且在VE2基因的上游插入7S,在VE3和VE4基因上游分别插入2S,来调控这3个基因在种仁特异表达。然后从这3个载体中酶切出分别带有启动子、基因和终止子的完整表达单元,构建到植物表达载体pCAMBIA1300中,所构建成的植物表达载体分别命名为pCAMBIA1300VE2·7S、pCAMBIA1300VE3·2S和pCAMBIA1300VE4·2S。测序分析显示3个基因的完整表达单元成功构建在植物表达载体pCAMBIA1300中。
3.为了更高效地改良花生中维生素E的合成效率,在单价载体的基础上构建了一个双价植物表达载体pCAMBIA1300 VE2·VE3,它含有VE_2和VE_3各自的启动子和终止子表达单元。为了同时改良花生中维生素E含量和油酸含量,亦构建得到双价植物表达载体pCAMBIA1300AT,它含有VE_4的完整表达单元和FAD2的反义RNA表达单元。
4、将pCAMBIA1300VE4·2S、pCAMBIA1300AT、pCAMBIA13002a等3个植物表达载体通过根癌农杆菌介导对花生进行遗传转化,提取抗性苗DNA进行PCR反应显示,他们的表达单元都已经插入到花生基因组中,各得到转基因苗3株、3株和6株,转化率分别为:2.00%、2.00%和3.85%。由于这些基因都是由种子特异表达启动子所引导,应待转基因苗种子收获后再作进一步检测。
综上所述,本研究构建了5个植物种子特异表达载体用来提高花生得维生素E含量,并将pCAMBIA1300VE4·2S、pCAMBIA1300AT、pCAMBIA13002a等3个载体对花生进行了转化,得到了转基因植株,为改良花生维生素E和油酸含量奠定了基础。
Peanut(Arachis hypogaea L.)is the world's fourth largest oilseed crop grown mainly in subtropical and tropical regions.China is the largest country of peanut production and consumption in the world.Vitamin E is an important class of lipid-soluble compounds with antioxidant activities that plays a very important role in plant,animal and human being.Plant oil is the main resource of the vitamin E in human nutritious.The content of activatedα-tocopherol is very low in plant,including peanut.To improve the rate of vitamin E component in peanut,we have tried to improve peanut vitamin E content by employing gene engineering.We reported here the cloning of homogentisic acid prenyltransferase(VE_2),2-methyl-6-phytylbenzoquinol(VE_3)andα-tocopherol methyltransferase(VE_4)genes,embryo-specific expression vectors construction and their transformation.
To make vitamin E genes express specifically in peanut embryo,two promoters already cloned in T-vectors were isolated with 99.8%and 100%identity to the sequences interested on GenBank,respectively.They are 7S seed storage protein gene promoter of soybean and 2S seed storage protein gene promoter of Brassica napus.The promoters region were 795bps and 1058bps in length.
Extracting total RNA from leaves of Arabidopsis thaliana and designing the primers according to the published sequence of VE_2,VE_3 and VE_4 genes on GenBank, we obtained those three genes,with 97%,100%and 100%identity to the conterpart sequences on GenBank,respectively,by RT-PCR.Those three genes were first constructed on intermediate expressing vector pSPROK with 7S promoter upstream to VE2 gene and 2S promoter upstream to VE3 and VE4 genes,respectively.Then the intact expression units with genes of interest flanked by specific promoters and nos terminals were cut and transferred to plant expressing vector pCAMBIA1300.Three plant expression vectors,named pCAMBIA1300VE2.7S,pCAMBIA1300VE3.2S and pCAMBIA1300VE4.2S,were obtained afterwards.Sequencing results showed that all the three genes were correctly inserted in plant expressing vector pCAMBIA1300.
To improve the tocopherol biosynthetic pathway and to increase the total amount of vitamine E,a plant bivalent expression vector,pCAMBIA1300 VE2.VE3,was constructed.It contains VE_2 and VE_3 genes,each has their own promoter and nos terminal.To improve both tocopherol and the rate of oleic acid component in peanut, another plant bivalent expression vector pCAMBIA1300AT was constructed.It has intact ultra-expression units of VE_4 and complete anti-RNA expression unit of FAD2.
Three vectors,pCAMBIA1300VE4.2S,pCAMBIA1300AT and pCAMBIA13002a, have been used to transform peanut variety Minghua 6,mediated by agrobacterium C58. Through many resistant transgenic seedling were obtained,only three,three and six seedlings with respective to genes of VE2,anti-RNA of FAD2 and VE4,and anti-FAD2 were shown to carry the genes of interest in the cells,after subjected to PCR assay,with transformation rate of 2.0%,2.0%and 3.85%.Further evaluation must be done after seed harvest,since those genes are all directed by seed specifically expressed prmoters.
In a word,we obtained five plant expressing vectors involved in increasing vatamine E contant.Plant expressing vectors pCAMBIA1300VE4-2S, pCAMBIA1300AT and pCAMBIA13002a were being used to transform peanut mediated by Agrobacterium and some transfomant seedlings were got,which provides basis in improving vitamin E and fatty acid composition in peanuts.
引文
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