VCAN及GRAMD3基因多态性及其与鸡胫黑色素沉着的关系研究

摘要

已有的研究推测VCAN及GRAMD3基因可能与黑色腿胫的形成有关，本研究的目的是研究Verican(VCAN)和Gram domain containing3(GRAMD3)基因及其与鸡胫色的关系。基于NCBI的数据，这两个基因分别位于五号染色体（Z染色体、即性染色体）长臂的14.3和23.2。本研究先利用DNA测序的方法在52只鸡的Versican和GRAMD3基因上分别寻找SNP，结果共找到12个突变位点，其中七个位于Versican、五个位于GRAMD3。分别对versican基因上的的内含子3(T161 C)，内含子4(G127 C)，内含子5(C143 T)，外显子7(G603 A)，外显子s8(G290A)，外显子14(G235 C)，外显子15(C154 T)以及GRAMD3基因上的外显子2(G199T)，内含子4(C81 G)，外显子6(G111 C)，内含子9(C128 T) and内含子11(A84G)进行分型。通过PCR-RFLP对多态进行检测，并用Z检验进行分析，然后利用SPSS对检验结果进行显著性分析。在这两个基因中，我们鉴定出GRAMD3第四外显子的非编码区的单尾分析结果为显著的P＜0.028717。该突变为无义突变，因此没有改变编码蛋白质氨基酸序列的编码区组成。但我们推测该突变可能是为鸡胫色的连锁标记或者通过其他方式影响鸡的胫色。

目录

声明

Contents

摘要

Abstract

Table of Abbreviation

1 Introduction

1．1 Why two genes Versican(Vcan)and GRAMD3 were selected to do studies in association with genes causing black shank color in the birds

1．1．1 Single nucleotide polymorphism

1．1．2 Characteristics of SNPS

1．1．3 Detection of SNPs

1．1．4 SSCP sequencing approach to detect SNP

1．1．5 Shot gun sequencing

1．1．6 Pyro sequencing

1．1．7 DNA Chip

1．2 Identification of Enzymes

1．3 SNP in the application of genetic diversity

1．3．1 Evaluation and conservation of genetic diversity

1．4 QTL Mapping and Marker-Assisted Selection

1．5 Population Genetic

2 Materials and Methods

2．1 Materials

2．1．1 Experimental Animal and Blood collection

2．1．2 Map

2．1．3 Fragments of VCAN and GRAMD3

2．1．4 Equipment，materials and sources

2．1．5 Reagents and sources

2．1．6 Commonly used reagents，plus the preparation

3 Method

3．1 Technical Experiment Scheme and Feasibility

3．1．1 Chickens DNA Genome Extraction through blood

3．1．2 Detection of DNA quality

3．2 Sex Identification

3．3 Primer Designing

3．4 PCR mix(100 reaction)

3．4．1 PCR Reaction VCAN gene

3．4．2 PCR Reaction for GRAMD 3 gene

3．5 Agarose Gel Electrophoresis

3．5．1 PCR reaction for the Chicken Versican gene

3．6 PCRreaction for GRAMD 3 gene

3．7 DNA Pooling

3．7．1 DNA Sequencing

3．7．2 Enzyme Mix and Digestion

3．8 SSCP PAGE

3．8．1 Staining solution

3．9 Genotyping with RFLP

3．9．1 SNP mapping

3．10 Statistical Analysis

4 Results

4．1 Extraction of Genomic DNA and their quality of Genomic DNA

4．2 Detection and Screening of SNP

4．3 Genotyping of selected SNPs

4．4 SNP screening

4．5 Association Analysis for VCAN and GRAMD3

4．6 Association of Genotypes with Phenotypes

4．6．1．Conservation of VCAN and GR AMD3 Genes

5 Discussion

6 Conclusion

Reference

ACKNOWLEDGEMENT