内蒙古白绒山羊KAP基因克隆、序列分析及PCR-SSCP检测

摘要

First，Cashmere goat DNA was extracted from the blood cell in this study，two pairs of primers，P 1／P2 and P3／P4，were designed according to the KAP gene sequences reported previously in other species．The result showed that two DNA fragments，S1 and S2，were amplified by PCR．The DNA fragments was ligated by T4 polymerase into the Sac Ⅱ and Not Ⅰ site of the cloning plasmid pGM-T Vector．And then，by performed the followingsteps：transfecting the competent TOP10，extracting the plasmid DNA from transfected cells，identilying restrictly the recombinant plasmids and sequence analysing，we obtained the Cashmere goat KAP coding sequences S1 and S2．S1 has 289 nucleotides，and S2 has 304 nucleotides．S2 and encodes a polypeptide containing 71 amino acids，respectively．Sequence Analysis indedicated．That the S2 fragment contains the Specif N-terminal structural domain and carboxy terminus structural domain that are highly conserved in the KAP6 gene family． Homogeneous analysis demonstrated that nucleotide sequences of Cashmere goat KAP gene between different species mammal are comparatively conservativeSNP(single nucleotide polymorphisms) is the most important research method in Molecular Genetics．First，the PCR—SSCP with about 300bp fragment have been established in the experiment．And then the present study selected KAP as a candidate gene to identify any difference in two gene fragments of KAP among Cashmere goat by PCR-SSCP．The results show that locus S2 in the genes(KAP6．1)which code the high-glycine—tyrosine keratin associated-protein，of keratin associated—protein family is significantly differential with cashmere yield(P<0．05)．Among the high—glycine-tyrosine keratin associated-protein，the AA and BB．genotypes of S2 are also significantly differential with cashmere yield(P<0．05)．The AB and BB genotypes of S2 are also significantly differential with cashmere fineness trait(P<0．05).

目录

英文文摘

声明

1引言

1.1山羊的主要品种及特征

1.1.1绒毛型山羊

1.1.2开士米型粗毛山羊

1.2绒山羊生产概况

1.3绒山羊育种研究进展

1.4分子标记在山羊遗传育种中的应用

1.4.1分子标记与山羊的起源、进化

1.4.2分子标记与山羊群体亲缘关系及群体遗传结构分析

1.4.3分子标记与山羊的重要经济性状

1.5角蛋白及其角蛋白关联蛋白

1.5.1角蛋白及角蛋白关联蛋白的种类

1.5.2角蛋白和角蛋白关联蛋白的结构

1.5.3毛发角蛋白及其角蛋白关联蛋白

1.5.4高甘氨酸/酪氨酸角蛋白关联蛋白

1.5.5角蛋白及其关联蛋白基因的研究进展

1.5.6角蛋白及其角蛋白关联蛋白功能的研究进展

1.6研究的目的和意义

2实验材料试剂及用品

2.1实验材料

2.2引物设计

2.3实验仪器和试剂

2.3.1主要仪器设备

2.3.2药品与酶

3实验方法

3.1绒羊基因组DNA的提取

3.2 PCR扩增反应

3.2.1引物处理

3.2.2最佳PCR反应条件的建立

3.2.3 PCR反应体系

3.2.4琼脂糖凝胶电泳检测PCR反应产物

3.3 PCR产物的克隆

3.3.1DNA片段回收

3.3.2克隆载体的构建

3.3.3 PCR产物与T载体连接(pGM-T Vector Systems)

3.3.4转化过程

3.3.5重组质粒的筛选与鉴定

3.3.6双酶切鉴定

3.4 DNA测序分析

3.5绒山羊KAP基因PCR产物多态性检测

3.6统计方法

3.6.1基因频率和基因型频率的计算

3.6.2遗传多态性分析

4实验结果与分析

4.1绒山羊基因组DNA的提取

4.2 PCR扩增结果

4.3 PCR产物的克隆

4.3.1阳性克隆菌落的PCR鉴定

4.3.2质粒的提取

4.3.3阳性克隆的双酶切鉴定

4.3.4 DNA测序结果

4.4绒山羊KAP基因序列碱基分布和限制性酶切分析

4.5绒山羊KAP基因cDNA序列与其他哺乳动物的同源性和进化分析

4.5.1绒山羊KAP1.3 cDNA序列及同源性和进化分析

4.5.2绒山羊KAP6.1 cDNA序列及其同源性和进化分析

4.6绒山羊KAP基因PCR-SSOP多态性检测结果分析

4.6.1标记基因和基因型与羊绒经济性状的相关性分析

4.6.2各个位点的基因频率和基因型频率

4.6.3各个位点的遗传参数值

4.6.4各个位点的基因和基因型与绒山羊经济性状关系分析

5讨论

5.1绒山羊基因组DNA的提取

5.2 PCR反应

5.3连接转化和克隆操作

5.4影响SSCP分析的因素

5.5抽样方法、样本容量和位点数量

6结论

致谢

参考文献

作者简介