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Journal of Zhejiang University (Agriculture and Life Sciences)  2020, Vol. 46 Issue (3): 376-382    DOI: 10.3785/j.issn.1008-9209.2019.06.211
Animal sciences & veterinary medicine     
Single nucleotide polymorphism and bioinformatics analysis of DQA2 gene in yak
Zhuo LI(),Lang CHEN,Tao JIANG,Lixia LIU,Li ZHANG,Rui WANG,Yaodong LI
College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China
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Abstract  

Single nucleotide polymorphism (SNP) of DQA2 gene in yak was analyzed via genomic DNA pooled amplification and directly sequencing, and further the influence of SNP sites on the secondary structures of mRNA and protein of DQA2 gene were predicted and analyzed by bioinformatic software. The results showed that the polymorphism of DQA2 gene in yak was high, and 14 SNP sites were detected. Only one SNP site was located on the intron 1, while 13 SNP sites were located on exon regions. Nine SNP sites were missense mutations, which resulted in changes in corresponding amino acids. The results of secondary structure prediction showed that eight SNP sites increased the stability of secondary structure of mRNA, and four SNP sites reduced the stability of secondary structure of mRNA. The SNP sites of missense mutation changed the secondary structure of DQA2 protein in yak. The proportion of random coil was the highest, followed by extended strand, and β-turn was the lowest among components of protein secondary structure before and after mutation. The results can provide basic data for further research on yak major histocompatibility complex (MHC) gene and theoretical basis for screening the molecular markers of yak disease resistance.



Key wordsyak      DQA2 gene      single nucleotide polymorphism site      bioinformatics     
Received: 21 June 2019      Published: 04 June 2020
CLC:  S 823.85  
Corresponding Authors: Zhuo LI     E-mail: smlz@xbmu.edu.cn
Cite this article:

Zhuo LI,Lang CHEN,Tao JIANG,Lixia LIU,Li ZHANG,Rui WANG,Yaodong LI. Single nucleotide polymorphism and bioinformatics analysis of DQA2 gene in yak. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(3): 376-382.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2019.06.211     OR     http://www.zjujournals.com/agr/Y2020/V46/I3/376


牦牛DQA2基因单核苷酸多态性及其生物信息学分析

利用基因组DNA混合池扩增产物直接测序的方法对55头牦牛DQA2基因的单核苷酸多态性(single nucleotide polymorphism, SNP)进行分析,并利用生物信息学软件预测分析了SNP位点对DQA2基因mRNA和蛋白质二级结构的影响。结果显示:牦牛DQA2基因多态性较高,共检测到14个SNP位点,仅有1个SNP位点位于内含子1上,其余13个均位于外显子区域,其中,9个SNP位点为错义突变,导致相应氨基酸发生改变。二级结构预测结果表明:8个SNP位点增大了mRNA二级结构的稳定性,4个位点降低了mRNA二级结构的稳定性。错义突变SNP位点均改变了牦牛DQA2蛋白质二级结构,突变前后二级结构各组分中无规则卷曲所占比例最高,其次为延伸链,β-转角的比例最低。研究结果为探究牦牛主要组织相容性复合体(major histocompatibility complex, MHC)基因的致病机制和免疫机制提供了有利条件,也为筛选牦牛抗病分子标记提供了理论基础。


关键词: 牦牛,  DQA2基因,  单核苷酸多态性位点,  生物信息学 

引物名称

Primer name

引物序列(5′→3′)

Primer sequence (5′→3′)

退火温度

Annealing temperature/℃

片段长度

Length of product/bp

P1

F: TACATCCCTCAACTGTGCACTC

R: GCTCTGCAACTCAGAACAGC

58.1153
P2

F: GAAGAGAAGCAGAATGGTGGAC

R: CCTGCTCCTCACCCTCAATTAT

58.1302
P3

F: CCAAAAGGGGTGGAACTCGT

R: GCGCCAAGCTTTTCCCATTT

59.6352
P4

F: ACCTTCTTTTCTAGGATGCGAC

R: CCAACATTCTGTATTCTGACTTTCA

55.9219
Table 1 Primer information of DQA2 gene
Fig. 1 Results of PCR amplification of DQA2 gene in yakM: 100 bp DNA marker; Lane 1-2: Amplification products of P1; Lane 3-4: Amplification products of P2; Lane 5-6: Amplification products of P3; Lane 7-8: Amplification products of P4.
Fig. 2 Sequencing peak diagrams of SNP sites for DQA2 gene in yak

SNP位点

SNP site

位置

Location

突变类型

Mutation type

氨基酸突变位点

Amino acid mutation site

等位基因频率 Allele frequency

突变前

Before mutation

突变后

After mutation

g.135T>C内含子10.620.38
g.4296A>G外显子2错义突变p.31I>V0.570.43
g.4374A>C外显子2错义突变p.57M>L0.700.30
g.5103G>A外显子3错义突变p.153V>I0.540.46
g.5127C>A外显子3错义突变p.161R>S0.600.40
g.5137T>C外显子3错义突变p.164L>P0.500.50
g.5141G>A外显子3同义突变0.660.34
g.5187C>T外显子3错义突变p.181P>S0.610.39
g.5527A>G外显子4同义突变0.510.49
g.5576G>C外显子4错义突变p.223G>R0.690.31
g.5602C>T外显子4同义突变0.730.27
g.5606G>A外显子4错义突变p.233V>M0.680.32
g.5659C>T外显子4同义突变0.800.20
g.5666C>A外显子4错义突变p.253P>T0.820.18
Table 2 Mutation information and allele frequency of SNP sites for DQA2 gene in yak
Fig. 3 Secondary structure of mRNA of SNP sites before and after mutation in yak DQA2 gene

SNP位点

SNP site

mRNA二级结构自由能

Free energy of secondary

structure of mRNA/(kJ/mol)

蛋白质二级结构 Secondary structure of protein/%

α-螺旋

Alpha helix

延伸链

Extended strand

β-转角

Beta turn

无规则卷曲

Random coil

突变前 Before mutation-1 077.0222.0530.317.8739.76
g.4296A>G-1 083.7223.6227.958.2740.16
g.4374A>C-1 092.5122.0528.747.0942.13
g.5103G>A-1 051.0723.6228.357.8740.16
g.5127C>A-1 080.7922.8328.748.2740.16
g.5137T>C-1 076.6020.8728.747.0943.31
g.5141G>A-1 068.6522.0530.317.8739.76
g.5187C>T-1 080.3724.8028.356.6940.16
g.5527A>G-1 082.8822.0530.317.8739.76
g.5576G>C-1 078.6925.9825.986.6941.34
g.5602C>T-1 080.7922.0530.317.8739.76
g.5606G>A-1 069.4921.6527.957.0943.31
g.5659C>T-1 093.3422.0530.317.8739.76
g.5666C>A-1 077.0223.6228.357.8740.16
Table 3 Free energy of secondary structures of mRNA and secondary structure of protein of SNP sites before and after mutation in yak DQA2 gene
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