【作者】 刘灵芝；

【导师】 王丕武；

【作者基本信息】 吉林农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 近年来，随着人民生活水平的提高以及对外贸易的开拓，作物营养品质研究日益引起人们的关注。利用基因工程技术改良作物营养品质的研究始于20世纪90年代，虽起步较晚，但通过近10年的发展，现已取得了一些可喜的成就。作物品质改良主要集中在改良种子贮藏蛋白、淀粉、油脂的含量和组成上。近年来国内外许多实验室竞相展开种子蛋白质基因工程研究。由于人类生产的植物蛋白主要来源于禾谷类和豆类作物的种子，故种子蛋白质的改良极为重要。改变贮藏蛋白质一直是禾谷类作物品质改良基因工程的研究课题。 大豆种子贮藏蛋白的含量远远高于其它作物，占种子干重的40％以上，是人类食物和动物养殖的主要植物蛋白来源。大豆蛋白氨基酸组分齐全，特别是含有全部8种人体必需氨基酸，同时还具有抗肿瘤、预防心血管疾病、增强免疫功能等生理保健功能。具有极高的营养价值和经济价值。大豆贮藏蛋白中大部分是球蛋白，主要包括大豆球蛋白(11S球蛋白)和β-伴大豆球蛋白(7S球蛋白)。11S球蛋白含硫氨基酸含量远高于7S蛋白，其含量比值(11S／7S)直接影响大豆蛋白营养品质及功能特性，从而影响大豆蛋白的应用价值。利用基因工程技术，筛选优良的大豆蛋白11S球蛋白基因资源，将其转移到同种或异种植物中，以提高其种子蛋白质含量、改良蛋白质品质，这在作物品质改良方面具有广阔的应用前景，为特殊蛋白质的生产开辟了道路。 本研究根据Genebank发袁的大豆11S球蛋白Gy5的cDNA序列，应用DNAstar生物软件设计了一对引物。用经过改良的畀硫氰酸胍法提取大豆未成熟种子的总RNA。用RT-PCR法扩增出目的片段，用EcoRI／PstI酶切后与pUC19载体连接，重组质粒用热激法转化大肠杆菌。穿刺培养的菌株送交宝生物工程有限公司测序，测序结果表明所扩增的片段符合预先设计，基因长度为1704bp，碱基准确率为99.6％。经氨基酸编码分析，有3个碱基为兼并予，其他3个碱基导致两个氨基酸有差异，氨基酸序列的同源性为99.6％。将此片段与具有CaMV35S启动子的表达载体pCAMBIA1301连接，构建了含有大豆贮藏蛋白基因Gy5的植物表达载体。用反复冻融法将质粒载体转入根癌农杆菌LBA4404中，用共培养法将Gy5导入烟草叶片，在已获得的再生植株中，经抗性选择和PCR检测获得了转化植株。 本研究克隆了一个优良种子贮藏蛋白质基因，初步证实了所构建的植物转化载体能够将大豆种子贮藏蛋白基因Gy5导入植物体并得到表达，为将其转移到同种或异种植物中，以提高其种子蛋白质含量和改善种子蛋白质品质的深入研究奠定了基础。吉林农业大学硕士论文大豆贮藏蛋白基因Gys的克隆及植物表达载体构建 此外，本研究对富含蛋白质、糖类等物质的组织或器官(如种子)的RNA提取方法进行了研究和改良。用改进后的方法所提取的RNA样品，经紫外分光光度仪测定，0D260/280=1 .9一2.0，OD260/23。>2.0，RNA提取率达0.05mg/g;经甲醛变性琼脂糖凝胶电泳，可看到285和185两条清晰的带，且前者带宽，说明RNA没有降解。此研究提高了RNA的提取效率和质量，建立了一种高效提取RNA的方法。 本研究还对RT一 PCR的条件从逆转录体系中模板RNA的用量、PCR体系中引物浓度、退火温度等几个方面进行了优化。确定在20ul逆转录体系中最佳模板RNA用量为1.sug PCR体系中最佳引物浓度为IPmol/ul;最佳退火温度为50℃。当用RT一PCR产物进行二次PCR时，可能是由于体系成分的改变，在退火温度为50℃时出现非特异带，所以对其退火温度又进行了摸索，最后确定为54℃，此时能扩增出专一的目的带。由此可以得出结论:在其它条件相同时，用逆转录产物做PCR的退火温度低于常规peR，比常规pCR低4oC左右。更多还原

【Abstract】 With the improvement of living standard and the development of foreign trade, people are paying more attention to improving nutritional quality of crops. At the beginning of 1990s, genetic engineering was used in this field and has got many achievements in recent ten years. These studies mostly focus on improving the content and component of storage protein, starch and lipid of seed.Crop seeds are the main source of plant protein for mankind, so improving the seed protein is very important. Soybean seeds contain more protein than any other cultivated commercial crops. Their storage protein accounts for over 40% of seeds dry weight. Soybean storage protein contains 8 kinds of human essential amino acids . It is an excellent source of dietary protein for people and animals. So soybean storage proteins have high nutritional value and commercial value.Globulin is the predominant seed storage protein in soybean, which is mainly composed of glycinin(HS) and B-conglycinin(7S). The content of sulfur-amino acid in glycinin is much higher than B-conglycinin, the content ratio(HS/7S)directly affects the quality and value of soybean storage protein. According to the above arguments, using genetic engineering technology clone the good soybean storage protein gene-US glycinin gene, then transfer it into the same or different plants to improve the storage protein content and quality, which has a good application prospect in the improvement of crop quality, and lays out a way for the production of special proteins.In this experiment, primers were designed according to the Gy5 cDNA sequence registered in Genebank. The total RNA of immature soybean seeds was extracted with improved Guanidine Thiocyanate method. The target fragment was amplified with RT-PCR and was cut by EcoRI/PstI, and ligated with pUC19 vector. This reconstructed plasmid was conducted into E.coli.DH5 a . The gene was sequenced and the results showed that the sequence contained 1704 nucleotides and only six nucleotides different from the Gy5 gene. Ligate this fragment with pCAMBIA1301, which is an expression vector with CaMV35s promoter. The recombinant was mobilized into Agrobacterium tumefaciens LBA4404 by the freeze-thaw method. Transform Gy5 into tobacco leaves. The Gy5 was integrated into acceptor, which was confirmed by resistant selection and PCR identification.This work cloned a good seed storage protein gene. It preliminarily confirmed that the reconstructed plant expression vector could make Gy5 integrated into acceptor. It also has laid on a foundation for further research on transferring the cloned gene into other plants in order to improve the content and quality of their seed storage protein.On the other side, the research has improved the isolated method of RNA from the plants with multitude of proteins, carbohydrates, et al. The extracted RNA was analyzed by ultraviolet spectrophotometer, the result was as follows: OD26o/28o=1.9~2.0, OD260/230>2.0; the content was 0.05mg/g. In the Agarose gel, 28S and 18S rRNA was recognized clearly and even the former was wider than the later. So this research improved the efficiency and quality of RNA extraction, it developed an efficient method for RNA extraction.At last, it was excellent to optimize RT-PCR in RNA quantity, primers density and annealing template in this research. It came to a conclusion: the optimum quantity is 1.5ug in 20ul reaction system; the optimum primers density is 1pmol/ul; the optimum annealing temperature is 50C. When using the products of RT-PCR as template proceed another PCR, the annealing temperature need be increased to 54C. The difference was probably caused because of the difference of the components in the system. So the RT-PCR annealing temperature was about lower 4C than normal PCR.更多还原