苦荞论文：苦荞蔗糖合酶的原核表达及纯化.docVIP

苦荞论文：苦荞蔗糖合酶的原核表达及纯化 【中文摘要】苦荞是一种具保健功能的作物,对于高血压和糖尿病有很好的改善作用,其富含的膳食纤维和荞麦蛋白能够减少肠道对于胆固醇的吸收、帮助中性脂排泄。蔗糖合酶是高等植物体内与蔗糖代谢密切相关的三类酶之一,它具有影响果实品质、参与细胞纤维生长和分化、参与淀粉合成及提高植物抗逆性等重要的生理功能。已有研究报道从马铃薯、甜菜、甘蔗等多种植物中克隆得到了蔗糖合酶,并进一步进行了该基因的调控与转基因研究,目前尚无从苦荞中克隆蔗糖合酶的相关报道。本研究在克隆苦荞蔗糖合酶基因的基础上,为进一步研究苦荞蔗糖合酶的生理特性和该酶在苦荞种子发育期的表达调控与籽粒中游离糖累积的关系以及制备该酶的多克隆抗体提供依据,本文开展了如下研究:（1）分析了从苦荞cDNA文库中克隆得到了蔗糖合酶基因序列,并对其编码的氨基酸序列进行了结构及功能预测、建立了系统发育树。结果表明:该基因序列包括1473bp的读码框（ORF）,编码含有490个氨基酸残基、分子量与等点电分别为53.4kD和6.02、N端含有28个氨基酸信号肽的蛋白质,该蛋白质的氨基酸序列与甜菜的蔗糖合酶氨基酸序列有100%的相似性,且系统发育树显示其与甜菜的亲缘关系最近,二级结构与三级结构预测结果表明其是由α-螺旋和β-折叠组成的“碗状结构”,功能结构域预测结果显示其包含蔗糖合成位点（sucrosesynth）与糖基化转移位点（Glycostransf1）。（2）以从苦荞cDNA文库中克隆得到的蔗糖合酶基因重组质粒为模板,PCR扩增得到苦荞蔗糖合酶编码基因,并将其定向插入原核表达载体pET28a构建了重组质粒pET28a-SuSy,转入大肠杆菌BL21 Star（DE3）进行表达,并对其表达条件进行优化。结果表明:其以包涵体形式在大肠杆菌中高效表达,优化的表达条件为:37℃,6h,IPTG终浓度为1.0mmol/L。Bandscan5.0软件检测显示其表达量为菌体蛋白的39.5%。（3）对重组蛋白进行了包涵体纯化、钴柱亲和层析及His-tag鉴定,结果显示该蛋白是N端带有His-tag的单一条带的蛋白。（4）用不同条件对以包涵体形式表达的重组蛋白进行复性,但未得到已复性的蛋白。本研究首次采用原核表达体系成功地实现了蔗糖合酶的重组表达,建立了亲和纯化体系,为进一步制备该酶的多克隆抗体以及研究该酶的表达调控奠定了基础。 【英文摘要】With a health care function and abundant dietary fiber, tartary buckwheat can contribute to control hypertension and diabetes, reduce assimilation of intestinal to cholesterol and assist nutral lipid in excreting. Sucrose synthase （SuSy） is one of the enzymes closely related to sugar metabolism, which has extraodinarily important physiological function such as influence on fruit quality, participation in growth and differentiation of cells fibers, starch synthesis, and has capability of increasing stress resistance of plants. It is reported that SuSy gene has been cloned from several kinds of plants such as solanum tuberosum, sugar beet and sugarcane which was further studied about gene expression and regulation.So far it is not reported as to cloning of SuSy from tartary buckwheat （Fagopyrumtataricum （L.）Gaertn）.This study amis to lay foundation for revealation of physiological property of SuSy, relationship between its regulation effect in seed development and accumulati