【作者】 陈振；

【导师】 谷晓峰；

【作者基本信息】 山东大学 ， 细胞生物学， 2009， 硕士

【摘要】 Globotriose是一种具有重要价值的寡糖。在大肠杆菌中,在外源添加乳糖情况下,它的合成需要3个关键酶基因:(1)susA,编码Sucrose synthase(EC2.4.1.13);(2)galE,编码UDP-galactose 4-epimerase(EC 5.1.3.2);(3)lgtC,编码α-1,4-galactosyltransferase(EC 2.4.1.x)。在植物中,Globotriose因缺少lgtC酶和乳糖而不能被合成。lgtC基因,来自奈瑟氏脑膜炎球菌(Neisseria meningitidis),该酶催化从UDP-Gal上转移一个半乳糖残基到乳糖末端上。本工作以转lgtC基因的甜菜植株为材料,研究甜菜细胞糖组分的变化,探求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甜菜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-半乳糖的含量。人的乳糖合成酶由二亚基组成,其调节亚基为乳清蛋白(α-lactalbumin),由LALBA基因编码;催化亚基为β-1,4-galactosyltransferase(EC 2.4.1.22),由Gal-T1基因编码,催化从UDP-Gal上转移一个半乳糖残基到葡萄糖末端上合成乳糖。本工作拟以转lgtC基因的甜菜丛生芽为受体将LALBA、Gal-T1基因转入甜菜,探讨利用转基因甜菜合成乳糖、Globotriose的可行性。转lgtC基因甜菜糖分的分析首先用RT-PCR方法筛选出lgtC基因高效表达的甜菜转基因株系,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分析,结果显示转正义基因的株系无论叶中还是块根中的可溶性总糖含量均较野生型株系显著提高,相应的葡萄糖、果糖和蔗糖含量与野生型株系相比也均有不同程度的提高;转基因株系UDP-半乳糖含量与对照相比显著降低(P≤0.05);而转反义基因株系的可溶性总糖、UDP-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,向甜菜中转入来源于原核生物的lgtC基因未影响植株的生长发育和光合作用。转乳糖合成酶基因甜菜的获得从人的cDNA文库(Marathon-Ready cDNA)中克隆得到乳糖合成酶基因(LALBA基因、Gal-T1基因),构建双基因串联的植物表达载体并转化已有的转lgtC基因甜菜丛生芽,获得聚合三个基因的甜菜植株。建立起四个转lgtC正义基因株系的离体丛生芽体系,经过继代培养发现,4个株系L1、L2、L3、L4与WT都有丛生芽发生,诱导时间相差不大,其中株系L2不定芽的发生时间较早。培养6周后,不同株系的诱导率均大于86%。随着丛生芽块继代培养时间的延长,不同株系间出现差异。有的株系的单芽在继代培养过程中增殖迅速,发生次生芽较多,而有些株系的单芽生长虽快但较少发生次生芽。选取lgtC基因表达活性较高且次生芽增殖迅速的株系L2、L4的丛生芽,通过农杆菌介导法将乳糖合成酶基因引入甜菜,经PCR检测获得了聚合三个基因(LALBA、Gal-T1、lgtC)的甜菜植株。本论文以lgtC基因和从人cDNA文库中分离的乳糖合成酶基因为目标基因,利用甜菜细胞为受体,获得了转入单个或数个目标基因的植株,同时研究了转基因植株中糖代谢途径的改变对糖分积累、生长发育的影响,为探测甜菜细胞糖代谢途径调控机制的研究提供了适宜的材料,同时为利用甜菜作为生物反应器生产高附加值寡糖、特别是Globotriose等奠定了基础。更多还原

【Abstract】 Globotriose is a kind of oligosaccharides of great value.In E.coli,the synthesis of Globotriose from added lactose needs three key enzyme gene:(1) susA,encoding sucrose synthase(EC2.4.1.13);(2) galE,encoding UDP-galactose 4-epimerase(EC 5.1.3.2);(3) lgtC,encodingα-1,4-galactosyltransferase(EC 2.4.1.x).Globotriose can not be synthesized in plants,for the lack of both lgtC enzyme and the substrate of lactose.lgtC gene,which comes from Neisseria meningococcus(Neisseria meningitidis), catalyzes the transfer of a galactose residue from UDP-Gal to the end of lactose.In this study,the variation of carbohydrate composition was analyzed in transgenic line containing lgtC,to explore the mechanism of expression of lgtC on metabolism, growth,and development of transgenic plants.In order to determine the constituent and content of soluble carbohydrates in the leaves of transgenic sugar beet accurately,various carbohydrates in transgenic plants were separated and identified by Thin Layer Chromotography(TLC).Meanwhile,the content of sucrose,glucose and fructose were measured with HPLC-RID,and the content of UDP-Gal was measured with RP-HPLC combining with PDAD.Lactose synthase has two subunits:whey protein(α-lactalbumin),the regulatory subunit,encoded by gene LALBA;andβ-1,4-galactosyltransferase(EC 2.4.1.22),the catalytic subunit,encoded by Gal-T1 gene,which catalyzes the transfer of a galactose residue from UDP-Gal to the end of glucose for the synthesis of lactose.The objective of my work is to transfer both LALBA and Gal-T1 gene into multiple bud of beet containing lgtC to obtain transgenic lines with all these transgenes mentioned above, to explore the feasibility of using transgenic sugar beet to synthesize lactose and Globotriose.Analysis of carbohydrate composition of lgtC transgenic beet plantsIn 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.The results suggested that the contents of total soluble carbohydrates in sense transgenic lines were significantly higher than that of WT,either in leaves or roots.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 the sense transgenic 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 were not significantly different compared with WT.Moreover,transgenic lines showed normal growth and photosynthesis compared with WT,suggesting that transferring lgtC gene from prokaryotes into sugar beet did not affect the growth and photosynthesis.Obtaining beet containing lactose synthase geneLactose synthase genes(both LALBA and Gal-T1) were cloned from human cDNA library(Marathon-Ready cDNA),and recombined to a plant expression vector and transferred into sugar beet containing lgtC,and transgenic lines containing LALBA,Gal-T1 and lgtC were obtained.Firstly multiple bud induction and subculture system of four lgtC transgenic lines and WT were established.Of the four lines,Line 2 and 4 had higher lgtC enzyme activity and more vigorous proliferation of secondary buds.LALBA and Gal-T1were transferred to buds of Line 2 and 4 by Agrobacterium-mediated transformation.PCR results suggested that plants pyramiding LALBA,Gal-T1,and lgtC were obtained。The transgenes expression and the soluble carbohydrate composition in the transgenic plants were to be determined. In this study,transgenic lines containing lgtC gene and and transgenic plants pyramiding lgtC and lactose synthase gene from cDNA library were produced and the change of soluble carbohydrate composition in transgenic plants and the impact of genetic modification of carbohydrate metabolic pathways on carbohydrate accumulation and growth,development of transgenic plants were also studied.The preliminary research provided some productive and useful lines for further study on the controlling mechanism of carbohydrate metabolism in sugar beet cells,and shed light on the insight into the use of engineered sugar beet as a bioreactor for production of oligosaccharides of high value,such as Globotriose.更多还原