白额高脚蛛毒腺cDNA文库构建..doc

白额高脚蛛毒腺cDNA文库构建 摘 要：蜘蛛的种类繁多且蜘蛛毒素成分具有多样性和多功能性，蜘蛛毒素已在神经生物学、分子药理学和分子毒理学等领域得到广泛应用；并且在临床和农业中也有着广泛的应用前景。随着基因工程和分子生物学的飞速发展，在农业方面培育抗虫转基因作物为害虫防治提 ... p摘nbsp; 要：蜘蛛的种类繁多且蜘蛛毒素成分具有多样性和多功能性，蜘蛛毒素已在神经生物学、分子药理学和分子毒理学等领域得到广泛应用；并且在临床和农业中也有着广泛的应用前景。随着基因工程和分子生物学的飞速发展，在农业方面培育抗虫转基因作物为害虫防治提供了一条新的途径。本实验研究的白额高脚蛛是一种大型室内蜘蛛，毒素具有对昆虫的专一性，而对人类没有伤害，可以作为抗虫基因的来源。本文介绍了蜘蛛毒腺如何取材，并通过利用真核生物mRNA 5 端的甲基化G (m7G)，5 端三磷酸键连接的特殊的帽子结构和3 端的Poly A尾的特点设计锚定引物。分别进行第一链合成，引物延伸合成第二链，酶切消化和分级分离柱回收cDNA，从而实现富集全长cDNA的目的。再将dscDNA与载体连接，然后将连接产物进行电转化，将连接产物电转到DH5alpha;感受态细胞后进行氯霉素筛选培养，选择转化效率最高的电转产物扩增培养。最后随机选取阳性克隆进行单菌落PCR分析，并电泳检测插入片段的大小，从而鉴定文库质量。br / 关键词：白额高脚蛛；毒素；cDNA文库；抗虫基因br / br / Construction of the cDNA Library from Brown Huntsman Spider Venom Glandbr / Abstract: Spider is a great variety and spider venom contains a rich cocktail of toxins, spider toxins which are widely used in neurobiology, molecular pharmacology and molecular toxicology and that have received increased attention from in clinical and agriculture application. With the rapid development of the technologies of gene engineering and molecular biology, to cultivate pest-resistant GM seeds have provided new ways to control insect pests. Heteropoda venatoria is a large-scale indoor spider whose toxin is only harmful to insect and breed out any diseases to humans. Thus these kinds of spiders can be used as the resource of anti insect gene. This article describes how to remove the spider venom, and by utilize methylation G (m7G) of eukaryotic mRNA5 ending and the specific cap sequence connected by the end of 5 three phosphate bond and the feature ofnbsp; 3 endings poly A tail to design anchor primer. Than respectively compound the first strand of cDNA, make use of the way of primer extension compound the second strand, digestion and recover cDNA, thus achieve the goals that enriching the whole cDNA. Then connecting dscDNA to vector, next use the products were to electroporation, then chloromycetin have been cultured and choose the best transformation efficien