果糖基转移酶融合基因转化玉米胚乳中果聚糖的积累
摘要
果聚糖是以β(2→1)和/或β(2→6)糖苷键连接的线性或分支型果糖聚合物,通常在末端连接一个葡萄糖残基,是水溶性的非还原性多糖。果聚糖广泛存在于细菌、真菌以及超过40000种高等植物中。研究较多的是菊糖型果聚糖,是一个蔗糖以β(2→1)糖苷键与多个果糖相连的线性分子。
果聚糖作为一种功能性食品,不仅可以增强人体钙的吸收,还被用作低热量值的甜味剂、食品添加剂中的水溶性膳食纤维等,更重要的是,它不能被人体代谢吸收,可以作为一种益生素,因此成为近年研究的热点。
菊糖型果聚糖代谢中主要涉及两个关键酶:蔗糖-1-果糖基转移酶(1-SST,EC2.4.1.99)和果聚糖-1-果糖基转移酶(1-FFT,EC2.4.1.100)。1-SST催化蔗糖转变为蔗果三糖(DP=3),1-FFT负责糖链的延长,将蔗果三糖转变为高聚合度的果聚糖。
在前期研究中已将由莴苣(Lactuca sativa)中克隆得到的1-SST、1-FFT基因分别与玉米醇溶蛋白基因启动子结合,构建目标基因串联的植物表达载体,使用农杆菌介导的芽尖转化法转化玉米自交系,获得初步鉴定的转基因植株。在此基础上,本研究以T_2代转基因植株为材料,通过除草剂初步筛选和PCR检测鉴定转基因植株,进而通过RT-PCR筛选出目标基因表达较高的株系,通过连续自交获得转基因纯合系。Southern分析表明,在入选材料中编码这两个酶的基因在玉米基因组中是以单拷贝的形式存在。对转基因植株的生长发育进行系统观察,与未转基因对照植株相比,转基因植株未出现明显不同的变化。
对转基因表达强度较高的株系,使用TLC(薄板层析)分离和鉴定玉米籽粒可溶性糖提取液中的各种糖分,同时采用高效液相色谱结合示差折光检测器测定技术,对玉米籽粒糖提取液中的可溶性糖进行定量分析。结果表明,在玉米籽粒中合成了一定量的果聚糖,合成量在0.05～2.0mg/g范围。蔗糖、葡萄糖、果糖其他可溶性糖的含量与对照植株差异不明显。
对玉米籽粒的蛋白提取液进行酶活性测定,确定了最适底物及浓度为100mmol/L蔗糖,最适反应时间为3h。然后从3个转基因株系籽粒中分别提取酶蛋白,进行酶活性测定。结果显示,该提取液具有果糖基转移酶活性,能以蔗糖为底物,生成果聚糖。这表明向不合成果聚糖的植物导入1-SST和1-FFT基因能够合成高分子量果聚糖。
本研究通过对转基因植株糖组分的分析和酶活性的测定,证明转入玉米中的果糖基转移酶1-SST和1-FFT发挥了作用,得到了能在玉米胚乳中积累果聚糖的新种质。该工作对于研究植物糖代谢网络及其调控机制也有启示作用。
Fructans are linear or branched polymers of repeating fructose residues connected byβ(2→1) and/orβ(2→6) fructosylfructose linkages,optionally including one terminal glucosyl unit.They are water-soluble and non-oxidative.Fructans are widely distributed in nature occurring in bacteria,fungi and over 40,000 higher plant species.The best characterized fructan is the inulin type,which has linear fructose chains attached to sucrose withβ(2→1) fructosylfructose linkages.
Many important nutritional and functional characteristics are attributed to inulin-type fructans.They can increase calcium absorbance,act as a low calorie sweetener or be used as a water-soluble dietary fiber in food ingredients.More importantly,inulin is not metabolized by humans and animals and can act as a prebiotic.
It is generally believed that inulin-type fructan biosynthesis in plants occurs through the concerted action of two enzymes,1-SST(sucrose:sucrose 1-fructosyltransferase,EC2.4.1.99) and 1-FFT(fructan:fructan 1-fructosyltransferase,EC2.4.1.100).1-SST catalyzes the conversion of sucrose to the trisaccharide 1-kestose(DP3).The elongation of 1-kestose to fructan(of DP up to 200) is catalyzed by 1-FFT.
During previous work,a coexpression vector was constructed including 1-SST and 1-FFT separately promoted by the zein promoter,which introduced fructan biosynthesis pathway into maize endosperms.This vector was transformed into maize bud using agrobacterium-mediated method.On this founation,transformants were selected on bialaphos and PCR assay.Transgenic lines that overexpressed target genes were confirmed by RT-PCR.Southern blot analysis suggested that these two enzymes are probably encoded by single-copy gene.Plant growth and kernel development were not affected.
Transgenic lines that overexpressed target genes were chosen for determination of carbohydrate content,using TLC for separation and HPLC for precise quantitative analysis.Data showed that,transgenic maize plants produced inulin-type fructan in the endosperms,ranging from 0.05～2.0 mg/g kernels.The levels of glucose,fructose and sucrose in these transgenic lines were not significantly different from WT plants.
To test the enzyme activity of 1-SST and 1-FFT,protein extracts of transgenic maize kernels were incubated with 100 mmol/L sucrose solution at 27℃and 3 h,and produced some fructan.These results suggested that the protein extracts had some level of fructosyltransferase activity,they can use sucrose as substrate to pruduce fructan.
In this study,we confirmed that the 1-SST and 1-FFT transformed into maize plants were active through carbohydrate analysis and measurement of enzyme activity,which means that the complete plant fructan biosynthesis pathway has been cloned into maize.We had obtained new type of maize lines which can be used to produce functional food in the future.
果聚糖作为一种功能性食品,不仅可以增强人体钙的吸收,还被用作低热量值的甜味剂、食品添加剂中的水溶性膳食纤维等,更重要的是,它不能被人体代谢吸收,可以作为一种益生素,因此成为近年研究的热点。
菊糖型果聚糖代谢中主要涉及两个关键酶:蔗糖-1-果糖基转移酶(1-SST,EC2.4.1.99)和果聚糖-1-果糖基转移酶(1-FFT,EC2.4.1.100)。1-SST催化蔗糖转变为蔗果三糖(DP=3),1-FFT负责糖链的延长,将蔗果三糖转变为高聚合度的果聚糖。
在前期研究中已将由莴苣(Lactuca sativa)中克隆得到的1-SST、1-FFT基因分别与玉米醇溶蛋白基因启动子结合,构建目标基因串联的植物表达载体,使用农杆菌介导的芽尖转化法转化玉米自交系,获得初步鉴定的转基因植株。在此基础上,本研究以T_2代转基因植株为材料,通过除草剂初步筛选和PCR检测鉴定转基因植株,进而通过RT-PCR筛选出目标基因表达较高的株系,通过连续自交获得转基因纯合系。Southern分析表明,在入选材料中编码这两个酶的基因在玉米基因组中是以单拷贝的形式存在。对转基因植株的生长发育进行系统观察,与未转基因对照植株相比,转基因植株未出现明显不同的变化。
对转基因表达强度较高的株系,使用TLC(薄板层析)分离和鉴定玉米籽粒可溶性糖提取液中的各种糖分,同时采用高效液相色谱结合示差折光检测器测定技术,对玉米籽粒糖提取液中的可溶性糖进行定量分析。结果表明,在玉米籽粒中合成了一定量的果聚糖,合成量在0.05～2.0mg/g范围。蔗糖、葡萄糖、果糖其他可溶性糖的含量与对照植株差异不明显。
对玉米籽粒的蛋白提取液进行酶活性测定,确定了最适底物及浓度为100mmol/L蔗糖,最适反应时间为3h。然后从3个转基因株系籽粒中分别提取酶蛋白,进行酶活性测定。结果显示,该提取液具有果糖基转移酶活性,能以蔗糖为底物,生成果聚糖。这表明向不合成果聚糖的植物导入1-SST和1-FFT基因能够合成高分子量果聚糖。
本研究通过对转基因植株糖组分的分析和酶活性的测定,证明转入玉米中的果糖基转移酶1-SST和1-FFT发挥了作用,得到了能在玉米胚乳中积累果聚糖的新种质。该工作对于研究植物糖代谢网络及其调控机制也有启示作用。
Fructans are linear or branched polymers of repeating fructose residues connected byβ(2→1) and/orβ(2→6) fructosylfructose linkages,optionally including one terminal glucosyl unit.They are water-soluble and non-oxidative.Fructans are widely distributed in nature occurring in bacteria,fungi and over 40,000 higher plant species.The best characterized fructan is the inulin type,which has linear fructose chains attached to sucrose withβ(2→1) fructosylfructose linkages.
Many important nutritional and functional characteristics are attributed to inulin-type fructans.They can increase calcium absorbance,act as a low calorie sweetener or be used as a water-soluble dietary fiber in food ingredients.More importantly,inulin is not metabolized by humans and animals and can act as a prebiotic.
It is generally believed that inulin-type fructan biosynthesis in plants occurs through the concerted action of two enzymes,1-SST(sucrose:sucrose 1-fructosyltransferase,EC2.4.1.99) and 1-FFT(fructan:fructan 1-fructosyltransferase,EC2.4.1.100).1-SST catalyzes the conversion of sucrose to the trisaccharide 1-kestose(DP3).The elongation of 1-kestose to fructan(of DP up to 200) is catalyzed by 1-FFT.
During previous work,a coexpression vector was constructed including 1-SST and 1-FFT separately promoted by the zein promoter,which introduced fructan biosynthesis pathway into maize endosperms.This vector was transformed into maize bud using agrobacterium-mediated method.On this founation,transformants were selected on bialaphos and PCR assay.Transgenic lines that overexpressed target genes were confirmed by RT-PCR.Southern blot analysis suggested that these two enzymes are probably encoded by single-copy gene.Plant growth and kernel development were not affected.
Transgenic lines that overexpressed target genes were chosen for determination of carbohydrate content,using TLC for separation and HPLC for precise quantitative analysis.Data showed that,transgenic maize plants produced inulin-type fructan in the endosperms,ranging from 0.05～2.0 mg/g kernels.The levels of glucose,fructose and sucrose in these transgenic lines were not significantly different from WT plants.
To test the enzyme activity of 1-SST and 1-FFT,protein extracts of transgenic maize kernels were incubated with 100 mmol/L sucrose solution at 27℃and 3 h,and produced some fructan.These results suggested that the protein extracts had some level of fructosyltransferase activity,they can use sucrose as substrate to pruduce fructan.
In this study,we confirmed that the 1-SST and 1-FFT transformed into maize plants were active through carbohydrate analysis and measurement of enzyme activity,which means that the complete plant fructan biosynthesis pathway has been cloned into maize.We had obtained new type of maize lines which can be used to produce functional food in the future.
引文
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