关岭牛TBC1D7基因单核苷酸多态性筛查及生物信息学分析

doi:10.3969/j.issn.1004-1524.2022.07.07

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (7): 1402-1411.DOI: 10.3969/j.issn.1004-1524.2022.07.07

关岭牛TBC1D7基因单核苷酸多态性筛查及生物信息学分析

刘鹏程¹(), 张继¹, 邱淦远¹, 龚俞², 李雪松², 李维², 张依裕¹, 刘若余¹^,^*()

1.贵州大学动物科学学院,高原山地动物遗传育种与繁殖教育部重点实验室,贵州贵阳 550025
2.贵州省畜禽遗传资源管理站,贵州贵阳 550001

收稿日期:2020-11-17 出版日期:2022-07-25 发布日期:2022-07-26
作者简介:* 刘若余,E-mail: liury1963@163.com
刘鹏程(1996—),男,贵州遵义人,硕士研究生,从事动物遗传育种与繁殖研究。E-mail: 591118954@qq.com
通讯作者: 刘若余
基金资助:
贵州省农业动植物育种项目(黔农育专字〔2018〕016号);贵州省农业科技攻关项目(黔科合NY〔2015〕3004-3);贵州省科技厅农业科技攻关项目(黔科合支撑〔2019〕2360号)

Single nucleotide polymorphism screening and bioinformatics analysis of TBC1D7 gene in Guanling cattle

LIU Pengcheng¹(), ZHANG Ji¹, QIU Ganyuan¹, GONG Yu², LI Xuesong², LI Wei², ZHANG Yiyu¹, LIU Ruoyu¹^,^*()

1. Key Laboratory of Plateau and Mountain Animal Genetics and Breeding, Ministry of Education, College of Animal Science, Guizhou University, Guiyang 550025, China
2. Guizhou Province Livestock and Poultry Genetic Resources Management Station, Guiyang 550001, China

Received:2020-11-17 Online:2022-07-25 Published:2022-07-26
Contact: LIU Ruoyu

摘要/Abstract

摘要：

为探究关岭牛TBC1D7(TBC1 domain family,member 7)基因单核苷酸多态性(single nucleotide polymorphism sites,SNPs)对其生长性状的影响。以贵州关岭牛为试验对象,构建DNA混合池,采用PCR扩增后直接测序法对关岭牛TBC1D7基因进行SNPs检测,并对其进行生物信息学分析。结果显示:关岭牛TBC1D7基因蛋白质编码区(CDS)全长882 bp,编码氨基酸293个,形成了一种不稳定的可溶性蛋白。该蛋白中不存在跨膜区域且不存在信号肽,为非分泌蛋白。蛋白中存在6个潜在的N-糖基化位点,二级结构主要由α-螺旋和无规则卷曲构成;在关岭牛TBC1D7基因CDS区共发现了4个同义突变位点,分别为c.402T>C、c.414A>G、c.609C>T和c.648T>C。4个突变位点均导致关岭牛TBC1D7基因mRNA二级结构、自由能和基因频率发生变化。本实验筛查到关岭牛TBC1D7基因4个SNPs位点,表明关岭牛TBC1D7基因多态性丰富,为进一步研究TBC1D7基因变异和关岭牛生长发育性状的相关性提供理论基础。

关键词: 关岭牛, TBC1D7基因, 单核苷酸多态性, 生物信息学

Abstract:

In order to explore effects of single nucleotide polymorphism(SNPs)of TBC1 domain family,member 7(TBC1D7)gene on cattle growth traits, the Guizhou Guanling cattle was selected as experimental objects to construct DNA pools. After PCR amplification, direct sequencing method was used to detect SNPs of TBC1D7 gene in Guanling cattle. The results showed that the coding region of TBC1D7 gene of Guanling cattle was 882 bp in length, encoding 293 amino acids. An unstable soluble protein was formed. There was no transmembrane region and no signal peptide in the protein encoded by TBC1D7 gene, which is a non-secretory protein. There were six potential N-glycosylation sites in the protein, and the secondary structure was mainly composed of α-helix and random coil. Four synonymous mutation sites were found in the coding sequence (CDS) region of TBC1D7 gene in Guanling cattle, namely, c.402T>C, c.414A>G, c.609C>T and c.648T>C. All the four SNPs resulted in changes in mRNA secondary structure, free energy and gene frequency of TBC1D7 gene in Guanling cattle. In this study, four SNPs loci of TBC1D7 gene in Guanling cattle were screened. The results showed that the polymorphism of TBC1D7 gene in Guanling cattle was abundant, which provided a theoretical basis for further research on the correlation between the variation of TBC1D7 gene and the growth and development traits of Guanling cattle.

Key words: Guanling cattle, TBC1D7 gene, single nucleotide polymorphism, bioinformatics

中图分类号:

S834.89

刘鹏程, 张继, 邱淦远, 龚俞, 李雪松, 李维, 张依裕, 刘若余. 关岭牛TBC1D7基因单核苷酸多态性筛查及生物信息学分析[J]. 浙江农业学报, 2022, 34(7): 1402-1411.

LIU Pengcheng, ZHANG Ji, QIU Ganyuan, GONG Yu, LI Xuesong, LI Wei, ZHANG Yiyu, LIU Ruoyu. Single nucleotide polymorphism screening and bioinformatics analysis of TBC1D7 gene in Guanling cattle[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1402-1411.

图/表 14

表1 关岭牛TBC1D7基因引物信息

Table 1 Primer information of TBC1D7 gene in Guanling cattle

引物名称 Primer name	上游引物 Forward primer (5'→3')	下游引物 Reverse primer(5'→3')	产物长度 Product length/bp	退火温度 Annealing temperature/ ℃	扩增区域 Amplified region
P1	CCCTGACGCCGAGGTAAC	CGTCCTCCAGGAAGAAATCA	397	56	3'UTR-Exon1-Intron1
P2	TGGCGAAAATCACGGAAT	TCTGGGATGACTGAGAACCA	248	58	Intron1-Exon2-Intron2
P3	TCACGATCAGCATGAAAAGC	ACCAATGACCCCATCAATTT	197	58	Intron1-Exon3-Intron2
P4	CCCACCCCGGGTCTCTAT	AGAGTCAAGCCAAGGGTCAA	245	60	Intron1-Exon4-Intron2
P5	TCTCTTGCCTCCTTGAGTTG	TGGTAAGGGCATTTCCTCAG	250	54	Intron1-Exon5-Intron2
P6	TGAGTAATCTCTTCCCCTTCAATC	CAGGATTTTTCTGACTCTGTGC	226	54	Intron1-Exon6-Intron2
P7	TTTGAAACTTGAGCTGTTTAGGTG	TTCAAGTGAAGCAAAGCACA	227	58	Intron1-Exon7-Intron2
P8.1	TTTCAGAGCCTGGTCTGCTT	CCACCCTGCATCTAATGACC	938	62	Intron7-Exon8-5'UTR
P8.2	GGACTTGATGCTTTGCTGAA	GTATGGCATTTGGGATGGAC	783	58	Intron7-Exon8-5'UTR
P8.3	AGGTCCTTGTTGGTCATCCA	TTCTGGAGCCAGGACTCACT	478	62	Intron7-Exon8-5'UTR

图1 关岭牛TBC1D7基因PCR扩增产物检测结果 M,DL2000 DNA marker; 1~8.3,引物P1~P8.3的 PCR扩增产物。

Fig.1 PCR amplified products of TBC1D7 gene in Guanling cattle M,DL2000 DNA marker; 1-8.3,PCR products of primer P1-P8.3.

图1 关岭牛TBC1D7基因PCR扩增产物检测结果 M,DL2000 DNA marker; 1~8.3,引物P1~P8.3的 PCR扩增产物。

Fig.1 PCR amplified products of TBC1D7 gene in Guanling cattle M,DL2000 DNA marker; 1-8.3,PCR products of primer P1-P8.3.

表2 关岭牛TBC1D7基因突变位点等位基因频率估算

Table 2 Estimation of allele frequency of TBC1D7 gene mutation site in Guanling cattle

突变位点 Mutation site	等位基因频率Allele frequency
突变位点 Mutation site	突变前Before mutation	突变后After mutation
c.402T>C	0.609 2	0.390 8
c.414A>G	0.607 1	0.392 9
c.609C>T	0.5254	0.474 6
c.648T>C	0.523 8	0.476 2

图3 关岭牛TBC1D7基因mRNA 二级结构

Fig.3 mRNA secondary structure of TBC1D7 gene in Guanling cattle

表3 TBC1D7基因编码蛋白的氨基酸组成

Table 3 Amino acid composition of TBC1D7 encoding protein in Guanling cattle

氨基酸 Amino acid	数量 Number	比例 Frequency/%	氨基酸 Amino acid	数量 Number	比例 Frequency/%
Ala	16	5.5	Leu	35	11.9
Arg	14	4.8	Lys	23	7.8
Asn	6	2.0	Met	8	2.7
Asp	15	5.1	Phe	14	4.8
Cys	9	3.1	Pro	15	5.1
Gln	10	3.4	Ser	26	8.9
Glu	23	7.8	Thr	9	3.1
Gly	7	2.4	Trp	5	1.7
His	9	3.1	Tyr	11	3.8
Ile	14	4.8	Val	24	8.2

图4 关岭牛TBC1D7基因编码蛋白亲水/疏水性预测结果正值表示疏水,负值表示亲水。

Fig.4 Hydrophilic/hydrophobic predictions of TBC1D7 encoding protein in Guanling cattle The positive value means the hydrophobicity,negative means hydrophilicity.

图5 TBC1D7蛋白跨膜区域分析

Fig.5 Analysis of TBC1D7 protein transmembrane region

图6 关岭牛TBC1D7基因编码蛋白的信号肽预测

Fig.6 Signal peptide analysis of amino acid encoded by TBC1D7 gene in Guanling cattle

图7 关岭牛TBC1D7基因编码蛋白的N-糖基化位点预测

Fig.7 N-glycosylation site prediction of amino acid encoded by TBC1D7 gene in Guanling cattle

图8 TBC1D7基因编码蛋白二级结构预测 h,α-螺旋; e,延伸链; t,β-转角; c,无规则卷曲。

Fig.8 Secondary structure prediction of TBC1D7 gene encoding protein h,Alpha helix; e,Extended strand; t,Beta turn; c,Random coli.

图9 TBC1D7基因编码蛋白三级结构预测

Fig.9 Tertiary structure prediction of TBC1D7 gene encoding protein

图10 TBC1D7基因编码蛋白功能结构域预测分析

Fig.10 Secondary structural domain prediction analysis of TBC1D7 gene encoding protein

图11 关岭牛TBC1D7基因编码蛋白互作网络预测

Fig.11 Prediction of TBC1D7 gene encoding protein interaction in guanling cattle

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关岭牛TBC1D7基因单核苷酸多态性筛查及生物信息学分析

Single nucleotide polymorphism screening and bioinformatics analysis of TBC1D7 gene in Guanling cattle

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