摘要
来自大肠杆菌的galE基因,编码UDP-galactose-4-epimerase(EC 5.1.3.2),该酶在正常的代谢途径中催化由UDP-葡萄糖向UDP-半乳糖的可逆转换。来自奈瑟氏脑膜炎球菌的lgtC基因,编码α-1,4-galactosyltransferase(EC 2.4.1.44),该酶催化半乳糖残基从供体分子转移到受体分子上。本工作利用分别转galE基因和lgtC基因的烟草植株研究细胞糖组分的变化,探求galE基因和lgtC基因对转基因植株代谢和生长发育的影响。
为了准确测定转基因烟草叶片的可溶性总糖组成和含量,本工作利用薄层层析方法分离和鉴定转基因植株的各种糖分,同时采用高效液相色谱分离和示差折光检测器测定技术定量叶片提取液中的蔗糖、葡萄糖和果糖含量,采用反向高效液相色谱分离和光电二极管阵列检测器测定UDP-葡萄糖和UDP-半乳糖的含量。
转galE基因烟草植株糖分测定以及galE酶活性分析
本工作首先用RT-PCR方法筛选出galE基因高效表达的烟草转基因株系,然后对这些株系进行可溶性总糖、单糖和二糖以及核苷糖测定。在烟草酶提取液中加入底物UDP-葡萄糖后,反应液中出现产物UDP-半乳糖,即存在galE催化的反应。测定转基因植株的galE在不同条件下的酶活性,得出该酶在pH7-9之间具有活力,最适pH为7.5-8,最适酶反应温度约为20℃。转正义基因株系UDP-半乳糖的生成量是野生型对照和转反义基因株系的1.5-1.6倍。
转基因烟草植株的糖分分析显示,转正义基因的株系E+41和E+54的可溶性总糖含量较对照株系显著提高,相应的葡萄糖、果糖和蔗糖含量与对照相比均有不同程度的提高;E+41和E+54的UDP-半乳糖含量与野生型相比显著提高,差异达到极显著(P≤0.01);UDP-葡萄糖含量与野生型相比也显著提高。而转反义基因株系E-23的UDP-糖、可溶性总糖以及其它糖分含量均与野生型植株无明显差异。
转基因植株生长发育观察和光合作用测定表明,向烟草中转入来源于原核生物的galE基因影响植株的生长发育和光合作用。对烟草种子萌发过程和植株生长发育各个时期的形态特征进行观察统计,结果发现,转正义基因植株在生长势方面明显优于野生型对照植株。与野生型对照植株相比,转正义基因植株幼苗具有更长的根系,在旺盛生长期具有更大的叶长、叶宽和叶面积,成熟期具有更高的植株高度,而且开花期相对较早。转正义基因植株的净光合速率明显高于野生型对照植株的。转反义基因植株在长势、开花期和净光合速率上均与野生型无明显差异。
转lgtC基因烟草糖分的分析
本工作采用RT-PCR技术选择出lgtC基因高效表达的转基因烟草株系,然后进行糖分测定,研究原核生物lgtC基因的引入对烟草植株体内糖组分及其含量的影响。
糖分分析显示,转正义基因的株系C+27和C+43的可溶性总糖含量较野生型对照株系显著提高,其中葡萄糖、果糖和蔗糖含量与对照相比也有不同程度的提高;C+27和C+43的UDP-半乳糖和UDP-葡萄糖含量较野生型均显著降低。而转反义基因株系C-32的可溶性总糖含量及葡萄糖、果糖、蔗糖的含量均与对照植株差别不大,但UDP-半乳糖和UDP-葡萄糖含量与野生型相比均有降低。
本论文分析了分别转galE和lgtC基因的烟草植株糖组分的变化,开展了原核生物糖代谢基因在植物基因工程中应用的尝试,探讨了糖代谢途径的遗传修饰对植株糖分积累和生长发育的影响。不仅为深入研究植物细胞糖代谢的调控机制鉴定出适宜的材料,而且为细胞糖代谢网络及其调控机制的揭示奠定了一定的工作基础,有重要的参考价值。
galE gene, coming from Escherichia coli, encodes UDP-galactose-4-epimerase (EC 5.1.3.2), which catalyzes the reversible conversation of UDP-Glc to UDP-Gal in regular metabolic pathways. lgtC, which comes from Neisseria Meningitidis, encodesα-1,4-galactosyl-transferase (EC 2.4.1.44). This enzyme mediates the transfer of galactosyl residue from donor to acceptor molecules. In this study, the variation of carbohydrate composition of transgenic tobacco cells containing galE or lgtC were studied respectively, in order to reveal the impact of heterologous expression of galE and lgtC gene on the metabolism and growth of transgenic tobacco plants.
Various carbohydrates in transgenic plants were separated and identified by Thin Layer Chromotography (TLC) to determine the constituent and content of soluble carbohydrates in the leaves of transgenic tobacco accurately. Furthermore, the content of sucrose, glucose and fructose were measured with HPLC-RID, and the content of UDP-Glc and UDP-Gal were measured perfectly with RP-HPLC combining with PDAD.
Determination of carbohydrate composition and galE enzyme activity in galE transgenic tobacco plants
In this study, transgenic lines that overexpressed target gene, confirmed by RT-PCR, were chosen for determination of carbohydrate content, including total soluble carbohydrates, monosaccharides, disaccharides and nucleotide sugars. When substrate UDP-Glc were added to the crude protein extracts of transgenic lines, the prospective product of UDP-Gal was detected in the reaction mixture, implying the exist of the reaction catalyzed by galE. The galE activity of transgenic plants was measured under different conditions. The epimerase was active between pH 7and pH9 with optimum at pH 7.5-8, and the optimal reaction temperature of the epimerase was about 20℃. The yield of UDP-Gal in the sense transgenic lines were significantly higher, usually 1.5-1.6 times as that of the antisense transgenic and WT lines.
The content of total soluble carbohydrates in sense transgenic lines (E+41, E+54) was significantly higher than that of WT. Accordingly, the levels of glucose, fructose and sucrose in these sense transgenic lines were all increased in considerable degrees. The content of UDP-Gal in sense transgenic lines (E+41, E+54) were extremely (P<0.01) higher than that of WT, while the content of UDP-Glc in the two lines were significantly (P<0.05) higher than that of WT also. However, the content of UDP-sugars, total soluble carbohydrates and other carbohydrate composition in the antisense transgenic line E-23 were not significantly different compared with WT plants.
Observation of growth and measurement of photosynthesis of transgenic lines suggested that the heterologous expression of galE from prokaryote activated the growth and photosynthesis of transgenic tobacco plants. The sense transgenic plants had better growth potential than WT plants. They had longer root system in seedling period, much leaf area in vigorous growing period due to longer and wider leaf, and higher plant height in maturation period, most interesting, the initiation of blossom was much earlier than WT plants. Additionally an obvious increase in net photosynthesis rate had been observed in sense transgenic plants, compared with WT plants. On the other hand, the growth potential, initiation of blossom and net photosynthesis rate of the antisense transgenic plants were not significantly different from WT plants.
Analysis of carbohydrate composition of lgtC transgenic tobacco plants
Transgenic lines with overexpression of lgtC gene, confirmed by RT-PCR, were chosen for determination of carbohydrate content, to reveal the effect of lgtC from prokaryote on the composition and content of carbohydrate in transgenic tobacco plants.
Total soluble carbohydrates of the sense lines (C+27, C+43) were significantly higher than that of WT. The analysis of carbohydrate composition of transgenic tobacco plants indicated that the levels of glucose, fructose and sucrose in these sense lines were increased to some extent. The content of UDP-Gal and UDP-Glc in the two lines were all significantly (P<0.05) lower than that of WT. However, the content of total soluble carbohydrates, as well as glucose, fructose and sucrose in the antisense line C-32 were not significantly different compared with WT plants, while the content of the UDP-Gal and UDP-Glc decreased generally in the antisense line C-32.
In this study, the variation of carbohydrate composition in galE or lgtC transgenic tobacco plants were investigated, respectively, and the impact of genetic modification of carbohydrate metabolic pathways on carbohydrate accumulation and growth of transgenic plants were also studied, with an attempt to utilize genes from prokaryote involved in carbohydrate metabolism to plant genetic engineering. This research not only provided some productive and useful lines for further study on the controlling mechanism of carbohydrate metabolism in plant cell, but also shed light on the insight into the cellular carbohydrate metabolism network and the controlling mechanism in plant cells, with great potential in the near future.
引文
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