摘要磁珠富集法是一种能够快速、高效地分离微卫星分子标记的方法。在本研究中，利用该方法分离狗脸水蛇(Cerberus rynchops)的微卫星分子标记。

通过常规的方法从狗脸水蛇的肌肉组织中提取大片段的基因组DNA，用限制性内切酶 Mse I进行酶切，回收纯化介于400-900bp的片段，用T4连接酶连上接头，构建一个“基因组PCR文库”。然后用带有生物素标记的微卫星探针(AC)10与其杂交，进行筛选。将杂交复合物结合到有链霉亲和素包被的磁珠上，经一系列的洗涤过程，去除磁珠表面不含微卫星的片段。随后将吸附在磁珠上的这些序列通过 PCR 扩增放大，连接PMD 19-T载体，将其转化入感受态大肠杆菌(Escherichia coli)DH 5α中，得到一个微卫星序列文库。76283

在本次研究中，在从狗脸水蛇中富集的基因组文库中分离出八个多态性微卫星分子标记。本研究所用的115个样本个体从新加坡收集而来。这些标记显示了高度的遗传多样性（每个位点有5-14个等位基因）和杂合度（HO介于0。565至0。965，HE介于0。701至0。882）。所有位点未显著偏离哈迪-温伯格平衡。同样并未得到任何一对基因座之间的连锁不平衡的证据。本次筛选的狗脸水蛇微卫星位点可应用于狗脸水蛇遗传多样性的检测及遗传图谱构建、物种资源的保护等研究领域。

Abstract Enrichment by magnetic beads is a simple and efficient method for rapid isolation of microsatellite DNA。 In this study, I isolated microsatellite DNA from dog-faced water snakes ( Cerberus rynchops ) genome with this method。

Genomic DNA was extracted from muscle tissue by normal methods and then digested with restriction enzyme Mse I。 Fragments of 400-900bp were isolated and purified and then ligated with short linkers (20bp) by ligase T4。 A whole genome PCR library was created。 This genomic DNA was hybridized with a biotin-labeled SSR (simple sequence repeat) probe (AC)10。 The hybrid mixture was incubated with magnetic beads coated with streptavidin。 After washing to remove the non-SSR fragments, the eluted single-stranded DNA contained the selected microsatellite DNA was obtained。 The selected DNAs were then amplified using primers designed complementary to the linkers, cloned into the PMD 19-T vector and sequenced into competent Escherichia coli DH 5α。

Here, eight polymorphic microsatellite loci were isolated from an enriched genomic library of C。 rynchops。 One hundred and fifteen inpiduals were collected from Singapore。 These markers revealed a high degree of genetic persity (5–14 alleles per locus) and heterozygosity (HO ranged from 0。565 to 0。965, and HE ranged from 0。701 to 0。882)。 No locus exhibited significant deviations from Hardy–Weinberg equilibrium。 There was no evidence of linkage disequilibrium between any pair of loci。 This subject started the screening of microsatellite primers of dog-faced water snake,and these can be applied in detection of genetic persity of dog-faced water snakes and genetic mapping, species protection and other research resources by preparing microsatellite molecular markers of dog-faced water snakes。

毕业论文关键词：狗脸水蛇； 磁珠富集法； 微卫星标记； PCR引物； 多态性

Key words: Cerberus rynchops;  Enrichment by magnetic beads;  Microsatellite markers;  PCR primers;  Polymorphism

目录

目录 4

引言 5

1  材料与方法 6

1。1  实验材料 6

1。2  试剂与仪器 6

1。3  实验方法 6

1。3。1 基因组DNA提取