【作者】 张成；

【导师】 王喆之；

【作者基本信息】 陕西师范大学 ， 植物学， 2006， 硕士

【摘要】 钙调蛋白(Calmodulin)是一种小分子酸性、耐热蛋白，等电点在3.9～4.3之间。钙调蛋白普遍存在于真核生物中，它的结构比较保守、变异小，在钙离子信号系统中起重要作用。钙调蛋白生理功能涉及面广泛：酶活性调节、激酶反应、细胞分裂与分化、细胞骨架与细胞运动、细胞分泌和渗透调节、光合作用、光形态建成、离子吸收转运、植物生殖生育、激素反应、细胞膜通道及基因表达调控等方面。迄今为止已成功地克隆了大麦、大豆、甘蓝、拟南芥、玉米等植物的钙调蛋白基因。 丹参为唇形科多年生草本植物，是我国的一种传统中药材。它在多方面有药用作用：预防心、脑血管疾病、抗肿瘤、抗炎症、抗病原微生物、抗氧化、抗衰老、促进修复再生等。我国数千年来应用传统中药治疗预防疾病，中草药是中华民族知识宝库中的重要组成部分。豇豆是一种常用蔬菜，其营养价值很高，含多种蛋白质、糖类、磷、铁、钙、和维生素及尼克酸，食物纤维等营养成分。丹参和豇豆钙调蛋白基因的克隆均未见报道。 本研究从丹参和豇豆叶片中提取总RNA。使用四种方法提取丹参总RNA：异硫氰酸胍法、CTAB硼砂法、SDS酸酚法、总核酸提取法。使用异硫氰酸胍法提取豇豆总RNA。通过比较各种提取丹参总RNA的方法，发现使用异硫氰酸胍法不仅方法简单、步骤较少，而且得到的丹参总RNA质量较高、产量较大，可以成功的应用于下游分子生物学实验。讨论了在不同条件下提取植物总RNA的影响因素，得出以下结论：研磨过程应适当增长，并保证植物材料浸泡在液氮中；整个实验过程应尽量在低温下进行，如果实验要求高温，也应适当减少植物总RNA处于高温的时间；多次乙醇洗涤有可能造成RNA降解或流失；植物总RNA提取最好采用幼嫩叶片作为材料，避免使用含杂质较多的子叶、茎等部位。 本研究克隆了丹参(Salvia miltiorrhiza Bunge)、豇豆(Vigna更多还原

【Abstract】 Calmodulin is a kind of small molecular acid protein with heat resistance, pI:3.9-4.3. Calmodulin is highly conserved in eukaryotes and a multifunctional protein which has an important function in Ca²⁺ signal system. Calmodulin takes part in many aspects: enzyme activity regulation,kinases response,cell division and differentiation,cell lines and movement,cell secretion and infiltration, photosynthesis, photomo rphogenesis,ion absorption and transit,plant reproductive and fertility,hormone response,cell membrane channel and Gene expression regulation etc. By now, lots of calmodulin genes have been cloned from many species: barely,soybean,wild cabbage,arabidopsis thaliana,maize.Salvia miltiorrhiza Bunge,a perennial plant, is a traditional herb in China. It has lots of medicine effects: curing heart and cerebrovascular diseases,anti-tumor,resisting to inflammation,anti-microbiologicaldiease,protecting from oxidation、 slowing oldness,renewing etc. Traditional herbs, belonging to an important part of Chinese knowledge, are used for thousands of years in China. Vigna unguiculata （L.）Walp is a very useful vegetable for humans. Vigna unguiculata （L.）Walp contains lots of nutritions, such as proteins,sugars,phosphorous,iron,calcium,vitamins and food fibers. Calmodulin from Salvia miltiorrhiza Bunge and Vigna unguiculata （L.）Walp have been not reported.Total RNA was isolated from leaves of Salvia miltiorrhiza Bunge and Vigna unguiculata （L.）Walp. There are four methods used in RNA isolation from Salvia miltiorrhiza Bunge: the method of Guanidine thiocyanate> the method of CTAB 、 the method of SDS 、 the method of total Nucleotide acid isolation. Compared with other methods, the method of Guanidine thiocyanate was considered as a method with simple steps and the good result. By analyzing different factors in isolation, it is concluded that: the更多还原