玉米γ-生育酚甲基转移酶基因的分离及其功能分析
摘要
维生素E又称生育酚,仅由光合生物合成,可以清除生物体内的活性氧和自由基,是人体必需的一类脂溶性维生素,具有重要的抗氧化功能。每天食用一定量的维生素E可以起到增强机体免疫力、防衰老、防癌、防心脑血管疾病及预防或减慢某些慢性疾病的作用。在天然存在的8种生育酚中,α-生育酚的生物活性最高,且被人体优先吸收和利用,但人类维生素E的主要膳食来源-食用植物油中,α-生育酚水平普遍比较低,而其前体γ-生育酚的水平却比较高。
γ－生育酚甲基转移酶(γ-TMT)是天然维生素E合成途径中的关键酶之一,其表达及活性对决定植物维生素E的组成起着重要作用,它催化6,γ-生育酚分别甲基化生成β,a-生育酚。玉米油是一种高品质的食用植物油,它含有86%的不饱和脂肪酸,其中56%是亚油酸,人体吸收率可达97%以上；玉米油中还含有少量的辅酶,也具有抗氧化性能；玉米油所含的谷固醇及磷脂,有防止衰老的功效,可降低人体内胆固醇的含量,增强人体肌肉、心脏和血管系统的机能,提高机体的抵抗能力。
此外,本研究初期分别克隆了高油玉米115号、大豆(Glycine max)、模式植物拟南芥(Arabidopsis thaliana)、结球甘蓝(Oryza sativa)的γ-TMT基因的全长cDNA序列,进行原核表达,结果显示玉米γ-TMT体外比活最高。
鉴于上述背景,本研究旨在通过分离玉米γ-TMT基因,并对其进行功能分析,为研究γ-TMT基因在维生素E生物合成中的作用和以后通过基因工程方法提玉米油α-生育酚水平奠定基础,主要结果如下：
1.提取玉米总RNA,利用RT-PCR技术,从高油玉米115号中分离了γ-TMT的全长cDNA序列(1059bp)。
2.构建了γ-TMT基因的原核表达载体pET-sigMTMT,并将其转化大肠杆菌BL21,纯化得到原核表达的高活性γ-生育酚甲基转移酶。
3.构建了植物表达载体pBI121-MTMT,粘花法转化拟南芥,抗性筛选转基因拟南芥,获得7个纯系。
4.提取纯系转基因拟南芥的总蛋白,进行ELISA检测和Western blotting分析。ELISA结果表明3#株系的γ-TMT活性最高,为野生拟南芥的4.56倍,6#为4.41倍;Western blotting也得到深浅不一的目的条带,表明不同株系具有活性不同的γ-TMT。
5.分别提取7个纯系的转基因拟南芥总RNA,通过Northern blotting分析表明,γ-TMT基因在转基因拟南芥中均有表达。
6.用HPLC技术测定7个纯系的α-生育酚和γ-生育酚含量变化,转基因植株的α-生育酚与γ-生育酚比值都高于野生型,其中6#最高为7.67,是野生型的13.14倍。7.构建了玉米γ-TMT基因与GFP融合的植物表达载体pBI121-MTMT-GFP,通过叶盘转化法转化烟草,通过对转基因烟草的根部细胞观察发现12颗有GFP表达,并且在细胞核中表达最强,其次是细胞膜,但并不是在所有细胞的核中都表达。
Tocopherols, commonly known as vitamin E, are a class of lipid-soluble antioxidants synthesized only by photosynthetic organisms. Tocopherols are essential to human because they perform numerous critical functions including quenching and scavenging various reactive oxygen species and free radical. Daily vitamin E supplementation results in decreased risk for cardiovascular disease, cancer and caducity, aid in immune function, and prevents or slows a number of degenerative diseases. Among the naturally occurring tocopherols,α-tocopherol has the highest bioactivity and is preferentially absorbed and distributed throughout the body. However, in the main diet resource, edible oil,α-tocopherol is present only as a minor component, while its biosynthetic precursor,γ-tocopherol, is present at high level.
γ-tocopherol methyltransferase(γ-TMT) is one of key enzymes of tocopherol biosynthetic pathway, whose expression and activity are important for component of tocopherols, and catalyzes methylation ofγ-tocopherol and 8-tocopherol toα-tocopherol andβ-tocopehrol respectively. Zea mays oil is one kind of high-quality edible vegetable oil, which contains 86% of unsaturated fatty acid, and 56% is sub oleic acid among them, the absorptivity of human body may reach more than 97%; There is a few of coenzyme which also has the antioxidation function in Zea mays oil. What be contained sitosterol and phosphatide in the oil can slow down decrepitude, reduce cholesterin contents of human body, strengthen the function of muscle, heart, blood vessel, immune system.
In addition, we cloned high oil maize 115, soybean, Arabidopsis, oryza'full-length cDNA sequence ofγ-TMT gene respectively in the beginning of the working, and expressed them in E.coli. The results showed thatγ-TMT from Zea mays had the highest activity.
Taking into account the above previous studies, we isolatedγ-TMT gene from Zea mays and defined the function of the genes for studying theγ-TMT gene's effect in vitamin E biosynthesis approach and paving the way for improving a-tocopherol content of Zea mays oil by means of genetic engineering. The main results were as follows:
1. The full-length cDNA sequence of y-TMT gene was isolated by RT-PCR from Zea mays.
2. A recombinant prokaryotic expression vector, named pET-sigMTMT, was constructed. We obtained high levelγ-TMT activity in E.coli. BL21.
3. A recombinant plant expression vector, called pBI121-MTMT, was constructed and introduced into Arabidopsis thaliana. Homozygousγ-TMT gene transgenic lines were obtained.
4. ELISA showed thatγ-TMT gene activity of 3# was the highest, which was about 4.56 times of wild type's,6#'s was about 4.41 times of wild type's. The hybridization belts of target protein with different shade were visualized by western blotting.
5. The result of northern blotting showed thatγ-TMT gene was expressed in transgenic lines.
6. HPLC showed that the ratio ofα-tocopherol toγ-tocopherol was higher than wild type. The ratio of 6# was 7.76, and it was 13.22 times of wild type.
7. A recombinant vector pBI121-MTMT-GFP with GFP was constructed forγ-TMT location in plant cell and was transformed into the genome of tobacco by agro-bacterium infecting leaf tobacco. The green fluorescence was visibled in nucleolus and cell membrance of root cells. GFP expression was stronger in nucleolus than in cell membrance, but not all nucleolus had fluorescence.
γ－生育酚甲基转移酶(γ-TMT)是天然维生素E合成途径中的关键酶之一,其表达及活性对决定植物维生素E的组成起着重要作用,它催化6,γ-生育酚分别甲基化生成β,a-生育酚。玉米油是一种高品质的食用植物油,它含有86%的不饱和脂肪酸,其中56%是亚油酸,人体吸收率可达97%以上；玉米油中还含有少量的辅酶,也具有抗氧化性能；玉米油所含的谷固醇及磷脂,有防止衰老的功效,可降低人体内胆固醇的含量,增强人体肌肉、心脏和血管系统的机能,提高机体的抵抗能力。
此外,本研究初期分别克隆了高油玉米115号、大豆(Glycine max)、模式植物拟南芥(Arabidopsis thaliana)、结球甘蓝(Oryza sativa)的γ-TMT基因的全长cDNA序列,进行原核表达,结果显示玉米γ-TMT体外比活最高。
鉴于上述背景,本研究旨在通过分离玉米γ-TMT基因,并对其进行功能分析,为研究γ-TMT基因在维生素E生物合成中的作用和以后通过基因工程方法提玉米油α-生育酚水平奠定基础,主要结果如下：
1.提取玉米总RNA,利用RT-PCR技术,从高油玉米115号中分离了γ-TMT的全长cDNA序列(1059bp)。
2.构建了γ-TMT基因的原核表达载体pET-sigMTMT,并将其转化大肠杆菌BL21,纯化得到原核表达的高活性γ-生育酚甲基转移酶。
3.构建了植物表达载体pBI121-MTMT,粘花法转化拟南芥,抗性筛选转基因拟南芥,获得7个纯系。
4.提取纯系转基因拟南芥的总蛋白,进行ELISA检测和Western blotting分析。ELISA结果表明3#株系的γ-TMT活性最高,为野生拟南芥的4.56倍,6#为4.41倍;Western blotting也得到深浅不一的目的条带,表明不同株系具有活性不同的γ-TMT。
5.分别提取7个纯系的转基因拟南芥总RNA,通过Northern blotting分析表明,γ-TMT基因在转基因拟南芥中均有表达。
6.用HPLC技术测定7个纯系的α-生育酚和γ-生育酚含量变化,转基因植株的α-生育酚与γ-生育酚比值都高于野生型,其中6#最高为7.67,是野生型的13.14倍。7.构建了玉米γ-TMT基因与GFP融合的植物表达载体pBI121-MTMT-GFP,通过叶盘转化法转化烟草,通过对转基因烟草的根部细胞观察发现12颗有GFP表达,并且在细胞核中表达最强,其次是细胞膜,但并不是在所有细胞的核中都表达。
Tocopherols, commonly known as vitamin E, are a class of lipid-soluble antioxidants synthesized only by photosynthetic organisms. Tocopherols are essential to human because they perform numerous critical functions including quenching and scavenging various reactive oxygen species and free radical. Daily vitamin E supplementation results in decreased risk for cardiovascular disease, cancer and caducity, aid in immune function, and prevents or slows a number of degenerative diseases. Among the naturally occurring tocopherols,α-tocopherol has the highest bioactivity and is preferentially absorbed and distributed throughout the body. However, in the main diet resource, edible oil,α-tocopherol is present only as a minor component, while its biosynthetic precursor,γ-tocopherol, is present at high level.
γ-tocopherol methyltransferase(γ-TMT) is one of key enzymes of tocopherol biosynthetic pathway, whose expression and activity are important for component of tocopherols, and catalyzes methylation ofγ-tocopherol and 8-tocopherol toα-tocopherol andβ-tocopehrol respectively. Zea mays oil is one kind of high-quality edible vegetable oil, which contains 86% of unsaturated fatty acid, and 56% is sub oleic acid among them, the absorptivity of human body may reach more than 97%; There is a few of coenzyme which also has the antioxidation function in Zea mays oil. What be contained sitosterol and phosphatide in the oil can slow down decrepitude, reduce cholesterin contents of human body, strengthen the function of muscle, heart, blood vessel, immune system.
In addition, we cloned high oil maize 115, soybean, Arabidopsis, oryza'full-length cDNA sequence ofγ-TMT gene respectively in the beginning of the working, and expressed them in E.coli. The results showed thatγ-TMT from Zea mays had the highest activity.
Taking into account the above previous studies, we isolatedγ-TMT gene from Zea mays and defined the function of the genes for studying theγ-TMT gene's effect in vitamin E biosynthesis approach and paving the way for improving a-tocopherol content of Zea mays oil by means of genetic engineering. The main results were as follows:
1. The full-length cDNA sequence of y-TMT gene was isolated by RT-PCR from Zea mays.
2. A recombinant prokaryotic expression vector, named pET-sigMTMT, was constructed. We obtained high levelγ-TMT activity in E.coli. BL21.
3. A recombinant plant expression vector, called pBI121-MTMT, was constructed and introduced into Arabidopsis thaliana. Homozygousγ-TMT gene transgenic lines were obtained.
4. ELISA showed thatγ-TMT gene activity of 3# was the highest, which was about 4.56 times of wild type's,6#'s was about 4.41 times of wild type's. The hybridization belts of target protein with different shade were visualized by western blotting.
5. The result of northern blotting showed thatγ-TMT gene was expressed in transgenic lines.
6. HPLC showed that the ratio ofα-tocopherol toγ-tocopherol was higher than wild type. The ratio of 6# was 7.76, and it was 13.22 times of wild type.
7. A recombinant vector pBI121-MTMT-GFP with GFP was constructed forγ-TMT location in plant cell and was transformed into the genome of tobacco by agro-bacterium infecting leaf tobacco. The green fluorescence was visibled in nucleolus and cell membrance of root cells. GFP expression was stronger in nucleolus than in cell membrance, but not all nucleolus had fluorescence.
引文
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