【作者】 崔巍；

【导师】 赵惠贤；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 小麦(Trticum aestivum)是我国乃至世界的主要粮食作物之一,其种植面积约占谷物种植面积的30%左右。小麦面粉可以加工成各种不同的食品,是人类植物蛋白质的重要来源,因此,对小麦的研究一直是人们关注的热点。小麦贮藏蛋白中的高、低分子量谷蛋白亚基是决定小麦面粉品质优劣的重要因素。低分子量麦谷蛋白亚基(LMW-GS)是小麦种子中一类重要的贮藏蛋白,占面筋蛋白的40%。研究表明,低分子量麦谷蛋白亚基与面粉的加工品质密切相关,它与高分子量麦谷蛋白亚基(HMW-GS)共同决定着面筋强度。高、低分子量麦谷蛋白亚基通过分子间二硫键形成麦谷蛋白大聚合体(GMP);谷蛋白大聚合体的含量、组成和粒度分布影响面团的特性。麦谷蛋白亚基的类型和数量决定了大聚合体的大小和数量。因此,可以通过选择优质麦谷蛋白亚基,改变聚合体的组成,提高大聚合体的含量来改良面团的加工特性。本研究以本课题组构建的含小偃6号低分子量麦谷蛋白基因XYGluD3-LMWGS1的重组质粒pGEM-XYGluD3-LMWGS1为模板,利用PCR技术扩增出基因XYGluD3-LMWGS1的完整编码区,构建成酵母表达载体,电激法转化酵母,并对转化子进行目的基因XYGluD3-LMWGS1进行蛋白诱导表达研究,研究取得以下结果:1.通过PCR扩增得到低分子量麦谷蛋白基因XYGluD3-LMWGS1的完整编码区,构建了克隆载体pMD18-simple T-XYGluD3-LMWGS,通过酶切对克隆载体进行了鉴定和测序,证实所获得的基因编码区序列与克隆的XYGluD3-LMWGS1中的可读框序列完全一致。表明基因XYGluD3-LMWGS1基因已经克隆进PMD18-simple T中。2.将克隆载体pMD18-simple T-XYGIuD3-LMWGS和酵母表达载体pPIC3.5K用相同的限制性内切酶切割,回收目的片段,用T4 DNA连接酶将两者连接,筛选得到重组酵母表达载体,经酶切鉴定和测序,XYGluD3-LMWGS1基因已经正确连接到pPIC3.5K上。3.用电激法将重组酵母表达载体导入甲醇型酵母(Pichia.pasteoris)菌株GS115中,获得转化子,将转化子进行PCR筛选,得到酵母重组菌株。4.在以甲醇为唯一碳源的培养基上培养,对重组酵母菌株进行了诱导表达。经过SDS-PAGE电泳检测,未发现目的蛋白表达,对诱导表达条件和目的基因序列进行分析发现,目的基因中酵母非偏爱密码子占总密码子的42%,大量稀有密码子的使用可能导致翻译过程中出现瓶颈效应,以及诱导时的PH值,温度及蛋白水解酶等因素可能最终导致目的蛋白的不表达。更多还原

【Abstract】 Wheat is one of the most important crops in the world, and the plant area of wheat is about 30% of all crops. The flour can be machininged all kinds of foodstuff and it is one of the sources of proteins for mankind.Therefore on the study of wheat storage protein is one of the focuses in cereal science.Both high- and low-molecular-weight glutenin subunits are the important components of wheat seed storage. Triticum aestivum low-molecular-weight glutenins subunits (LMW-GS) is a kind of important storage protein and account for 40% of wheat gluten protein content. These proteins play a significant role on flour-processing properties. LMW and High-molecular-weight glutenins subunits (HMW-GS) are considered to significantly affect dough-quality characteristics. They incorporate in glutenin macropolymer (GMP) through intermolecular disulphide bonds. It was reported that the content、component and size distribution significantly affect the quality characteristics of wheat flour.The composition and quanity of glutenins affect the size distribution of glutenin macropolymer (GMP). So, wheat flour processing properties can be improved by enhancing the quanity of GMP and altering GMP component through introducing genes related to good quality into wheat cultivar.This reasearch amplified XYGluD3-LMWGS1 gene by PCR,and constructed the yeast expressive vectors.The expression of XYGluD3-LMWGS1 in Pichia.pasteoris were studied.The main achievements were as follows:1.XYGluD3-LMWGS1 gene was amplified from the pGEM-XYGluD3-LMWGS1,the XYGluD3-LMWGS1 gene was cloned into PMD18-simple T.The recombinants were assayed by the digestion of restricted enzyme and sequenced.The results indicated that the XYGluD3-LMWGS1 gene was cloned into PMD18-simple T.2.The clone vector and pPIC3.5K were digested by the restricted enzyme,the ligated by T4 DNA ligase.The XYGluD3-LMWGS1 gene was cloned into pPIC3.5K by analysis.3. The expressive vector was transformed into Pichia.pasteoris(GS115) by electricity dash method.Transformants were obtained by PCR.The recombinants were obtained bt the assay of PCR.4.The strain express the XYGluD3-LMWGS1 in the culture medium using methyl alcoholas the carbolic rescource.Through the SDS-PAGE,expressed pretein were not found.Analysis of the sequence of gene and the condition of culture were found,the optimal condon just 58% in all condon,not enough optimal condon maybe influence the efficiency of translation bring down.The PH,temperature and other conditions maybe influence the expression of protein.更多还原