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DH5α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。
关键词：白额高脚蛛；毒素；cDNA文库；抗虫基因

Construction of the cDNA Library from Brown Huntsman Spider Venom Gland
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 of  3' ending's 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 efficiency of the products were. Finally randomly selected positive clone was analyzed by the method of single colony PCR, and inserted fragment was checked the size by means of electrophoresis to test quality of the library.
    Key words: Heteropoda venatoria; toxin; cDNA library; Insect-resistant genes

2  材料与方法
2.1  材料与试剂
2.1.1  组织样品
白额高脚蛛采集于校园，并在大学基因工程实验室进行人工饲养一周。
2.1.2  质粒和菌株
质粒载体pNDR-LIB购自Clontech公司。大肠杆菌（Escherichiacoli）DH5α购自Invitrogen公司，由实验室保存，其基因型为：DH5α：supE44 Δlac U169（φ80lacZΔM15） hsdR17 recA1 gvrA96 thi-1relA1
2.1.3  RNA提取试剂
DEPCS水：每1L原子级水（＞18MΩ）中加入1mlDEPC，终浓度为0.1%，37ΟC摇床过夜混匀后，高压灭菌。

目    录   10000字
摘  要    1
关键词    1
1  前言    2
2  材料与方法    4
2.1  材料与试剂    4
2.1.1  组织样品    4
2.1.2  质粒和菌株    4
2.1.3  RNA提取试剂    4
2.1.4  缓冲液    4
2.1.5  培养基    5
2.1.6  试剂和酶    5
2.2  实验方法    5
2.2.1  缓冲液的配置    5
2.2.2  培养基的配置    6
2.2.3  其他溶液的配置    6
2.2.4  提取RNA的实验用品的处理    6
2.2.5  白额高脚蛛毒腺组织的处理    7
2.2.6  总RNA的提取与检测    7
2.2.7  磁珠法分离白额高脚蛛毒腺mRNA    7
2.2.8  白额高脚蛛cDNA的合成    8
2.2.9  dscDNA与载体的连接    9
2.2.10  白额高脚蛛cDNA文库的转化    10
2.2.11  质粒文库的扩增和保存    10
2.2.12  cDNA克隆PCR分析    10
3     实验结果与分析    11
3.1  白额高脚蛛毒腺总RNA的提取    11
3.2  cDNA的合成    11
3.3  白额高脚蛛毒腺cDNA文库的构建和鉴定    12
4  实验讨论与总结    13
4.1  限制性内切酶    13
4.2  RNA提取    13
4.3  电转化    14
4.4  实验总结    14
参考文献    14
致    谢    15