SNPs and Correlation of TYRP1b with Color of Zebrafish
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摘要:
目的 探究酪氨酸酶相关蛋白1b(tyrosinase-related protein 1b, TYRP1b)基因变异与斑马鱼体色性状的相关性。 方法 以体色表型存在显著差异的红色、黄色和蓝色斑马鱼共计219个样本的肌肉组织DNA为模板,设计3对引物,采用PCR扩增、Sanger测序技术筛查TYRP1b基因单核苷酸多态性(single nucleotide polymorphisms, SNPs),并将SNPs与斑马鱼体色性状进行关联分析。 结果 获得的斑马鱼TYRP1b基因外显子2、部分内含子3、外显子4、内含子4、外显子5、内含子5、外显子6和外显子7长度分别为134、212、168、135、180、113、150、171 bp。SNPs筛查显示,3种体色斑马鱼群体共筛查到18个SNPs,部分内含子3存在11个SNPs位点:g.2125G>A、g.2150G>A、g.2152G>T、g.2161C>A、g.2175A>T、g.2180G>T、g.2185A>T、g.2192A>C、g.2200A>T、g.2201A>C和g.2213T>C;内含子4检测到4个SNPs位点:g.7C>A、g.65A>G、g.84T>A和g.103C>T;内含子5存在1个SNPs位点:g.61G>T;外显子4、6分别存在1个SNPs位点:g.125G>T和g.80T>A,且均为同义突变;外显子2、5和7均未检测到SNPs。关联分析表明,TYRP1b基因内含子3中g.2152G>T、g.2175A>T、g.2180T>G、g.2192A>C、g.2200A>T、g.2201A>C位点、内含子4中g.65A>G位点及外显子6中g.80T>A位点的基因型均与斑马鱼体色性状极显著相关(P<0.01)。 结论 斑马鱼TYRP1b基因内含子3、4及外显子6存在8个与体色表型存在关联的SNPs,可能影响斑马鱼体色性状或与之紧密连锁。 -
关键词:
- 斑马鱼 /
- 酪氨酸酶相关蛋白1b基因 /
- 单核苷酸多态性 /
- 体色性状 /
- 关联分析
Abstract:Objective Correlation between the mutation sites of tyrosinase related protein 1b gene, TYRP1b, and body color traits of Danio rerio was investigated. Method DNA of muscles from zebrafish of three significantly different body color phenotypes were used as the templates. Single nucleotide polymorphisms (SNPs) of the genes were tested by PCR and Sanger direct sequencing to determine the relationship between the mutation sites of TYRP1b and traits of zebrafish body color. Result The exon 2, partial intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, and exon 7 of TYRP1b were 134 bp, 212 bp, 168 bp, 135 bp, 180 bp, 113 bp, 150 bp, and 171bp in length, respectively. Eighteen SNPs were found in 219 zebrafish with body color of red, yellow, and blue. Of them, 11 (i.e., g.2125G>A, g.2150G>A, g.2152G>T, g.2161C>A, g.2175A>T, g.2180G>T, g.2185A>T, g.2192A>C, g.2200A>T, g.2201A>C, and g.2213T>C) were located in partial intron 3, 5 (i.e., g.7C>A, g.65A>G, g.84T>A, and g.103C>T) in intron 4 and g.61G>T in intron 5, and the synonymous mutation g.125G>T in exon 4 and g.80T>A in exon 6. No SNPs were detected in exons 2, 5, and 7. An association analysis showed that the genotypes of g.2152G>T, g.2175A>T, g.2180T>G, g.2192A>C, g.2200A>T, and g.2201A>C in intron 3, g.65A>G in intron 4, and g.80T>A in exon 6 of TYRP1b significantly correlated with zebrafish body color (P<0.01). Conclusion It appeared that 8 SNPs in introns 3, 4, and exon 6 of TYRP1b in D. rerio were associated with body color phenotype of the fish. -
Key words:
- Danio rerio /
- TYRP1b /
- single nucleotide polymorphism /
- body color trait /
- correlation analysis
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图 1 斑马鱼TYRP1b基因3对引物PCR产物电泳图谱
M:DL2000 DNA Marker;A图中1~6:Tb-2引物PCR产物;B图中1~3:Tb-1引物PCR产物,4~6:Tb-3引物PCR产物。
Figure 1. Electrophoresis of products of TYRP1b in zebrafish from 3 pairs of primers
M: DL2000 DNA marker; A1-6: PCR product of Tb-2 primer; B1-3: PCR product of Tb-1 primer; B4-6: PCR product of Tb-3 primer.
表 1 引物信息
Table 1. Information on primer
引物
Primers序列(5′-3′)
Sequence(5′-3′)退火温度
Annealing temperature/ ℃延伸时间
Extended time/s产物长度
Product length/bp扩增区域
Amplified regionTb-1 F:CTGTTGTCTGGCCCGAAGAT 60.2 25 134 外显子2
Exon 2R:AGGAATGTGCTGACGCTGAG Tb-2 F:CCCACAGCTCTAATGCAAACT 59.6 59 956 部分内含子3~外显子6
Partial intron 3–exon 6R:GCAATCAGCCGTCACTCACTT Tb-3 F:GTAGCGTCACACCACTGACT 58.4 28 171 外显子7
Exon 7R:ATCACGGTAACGGTGGTAGC 表 2 不同体色斑马鱼TYRP1b基因内含子3中11个SNPs的基因型与等位基因频率
Table 2. Genotypes and allele frequencies of 11 SNPs in intron 3 of TYRP1b of zebrafish with different body colors
突变位点
SNPs基因型与等位基因
Genotype and allele基因型与等位基因频率
Genotype and allele frequencies红色 RZ 黄色 YZ 蓝色 BZ g.2125G>A GG 1.0000(70) 0.5625(36) 1.0000(85) GA 0(0) 0.3750(24) 0(0) AA 0(0) 0.0625(4) 0(0) G 1.0000 0.7500 1.0000 A 0 0.2500 0 g.2150G>A GG 0.2000(14) 1.0000(64) 0.7647(65) GA 0.4000(28) 0(0) 0.2353(20) AA 0.4000(28) 0(0) 0(0) G 0.4000 1.0000 0.8823 A 0.6000 0 0.1177 g.2152G>T GG 0.2000(14) 0.3125(20) 0.4706(40) GT 0.4000(28) 0.3750(24) 0.5294(45) TT 0.4000(28) 0.3125(20) 0(0) G 0.4000 0.5000 0.7353 T 0.6000 0.5000 0.2647 g.2161C>A CC 1.0000(70) 0.3125(20) 0.7059(60) CA 0(0) 0.3750(24) 0.2941(25) AA 0(0) 0.3125(20) 0.0000(0) C 1.0000 0.5000 0.8530 A 0 0.5000 0.1470 g.2175A>T AA 0.4000(28) 0.5000(32) 0.4706(40) AT 0.4000(28) 0.5000(32) 0.5294(45) TT 0.2000(14) 0(0) 0(0) A 0.6000 0.7500 0.7353 T 0.4000 0.2500 0.2647 g.2180T>G TT 0.2000(14) 0.3125(20) 0.4706(40) TG 0.4000(28) 0.3750(24) 0.5294(45) GG 0.4000(28) 0.3125(20) 0(0) T 0.4000 0.5000 0.7353 G 0.6000 0.5000 0.2647 g.2185A>T AA 1.0000(70) 0.3125(20) 0.7059(60) AT 0(0) 0.3750(24) 0.2941(25) TT 0(0) 0.3125(20) 0(0) A 1.0000 0.5000 0.8530 T 0 0.5000 0.1470 g.2192A>C AA 0.2000(14) 0.3125(20) 0.4706(40) AC 0.4571(32) 0.3750(24) 0.5294(45) CC 0.3429(24) 0.3125(20) 0(0) A 0.4286 0.5000 0.7353 C 0.5714 0.5000 0.2647 g.2200A>T AA 0.2000(14) 0.3125(20) 0.4706(40) AT 0.4571(32) 0.3750(24) 0.5294(45) TT 0.3429(24) 0.3125(20) 0(0) A 0.4286 0.5000 0.7353 T 0.5714 0.5000 0.2647 g.2201A>C AA 0.2000(14) 0.5000(32) 0.4706(40) AC 0.4571(32) 0.5000(32) 0.5294(45) CC 0.3429(24) 0(0) 0(0) A 0.4286 0.7500 0.7353 C 0.5714 0.2500 0.2647 g.2213T>C TT 0.2000(14) 1.0000(64) 0.7059(60) TC 0.4571(32) 0(0) 0.2941(25) CC 0.3429(24) 0(0) 0(0) T 0.4286 1.0000 0.8530 C 0.5714 0 0.1470 括号内的数字为SNPs位点基因型数,下表同。
Data in brackets are number of genotypes. Same for below.表 3 不同体色斑马鱼TYRP1b基因内含子4、5和外显子4、6中7个SNPs的基因型与等位基因频率
Table 3. Genotypes and allele frequencies of 7 SNPs of TYRP1b in introns 4, 5 and exons 4, 6 in zebrafish of different body colors
分布区域
Distribution region突变位点
SNPs基因型与等位基因
Genotype and allele基因型与等位基因频率
Genotype and allele frequencies红色 RZ 黄色 YZ 蓝色 BZ 内含子4
Intron 4g.7C>A CC 1.0000(70) 0.5625(36) 1.0000(85) CA 0(0) 0.3750(24) 0(0) AA 0(0) 0.0625(4) 0(0) C 1.0000 0.7500 1.0000 A 0 0.2500 0 g.65A>G AA 0.2000(14) 0.5625(36) 0.7059(60) AG 0.4571(32) 0.3750(24) 0.2941(25) GG 0.3429(24) 0.0625(4) 0(0) A 0.4286 0.7500 0.8530 G 0.5714 0.2500 0.1470 g.84T>A TT 1.0000(70) 0.3125(20) 0.7059(60) TA 0(0) 0.3750(24) 0.2941(25) AA 0(0) 0.3125(20) 0(0) T 1.0000 0.5000 0.8530 A 0 0.5000 0.1470 g.103C>T CC 0.2000(14) 0.7500(64) 0.7647(65) CT 0.4571(32) 0.0625(0) 0.2353(20) TT 0.3429(24) 0.1875(0) 0(0) C 0.4286 1.0000 0.8823 T 0.5714 0 0.1177 内含子5
Intron 5g.61G/T GG 1.0000(70) 0.5625(36) 1.0000(85) GT 0(0) 0.3750(24) 0(0) TT 0(0) 0.0625(4) 0(0) G 1.0000 0.7500 1.0000 T 0 0.2500 0 外显子4
Exon 4g.125G>T GG 1.0000(70) 0.5625(36) 1.0000(85) GT 0(0) 0.3750(24) 0(0) TT 0(0) 0.0625(4) 0(0) G 1.0000 0.7500 1.0000 T 0 0.2500 0 外显子6
Exon 6g.80T>A TT 0.4571(32) 0.5000(32) 0(0) TA 0.3429(24) 0.5000(32) 0.5294(45) AA 0.2000(14) 0(0) 0.4706(40) T 0.6286 0.7500 0.2647 A 0.3714 0.2500 0.7353 表 4 TYRP1b基因SNPs位点的各基因型分布及其与体色表型的关联分析
Table 4. Distribution and association between TYRP1b SNPs genotypes and body color phenotypes
分布区域
Distribution region位点
SNPs基因型
Genotype不同体色数量
Number of different body color总计
Totalχ2 P值
P value红色 RZ 黄色 YZ 蓝色 BZ 内含子3
Intron 3g.2152G>T GG 14 20 40 74 42.4995 <0.0001 GT 28 24 45 97 TT 28 20 0 48 g.2175A>T AA 28 32 40 100 32.0120 <0.0001 AT 28 32 45 105 TT 14 0 0 14 g.2192A>C AA 14 20 40 74 38.4456 <0.0001 AC 32 24 45 101 CC 24 20 0 44 g.2201A>C AA 14 32 40 86 61.2982 <0.0001 AC 32 32 45 109 CC 24 0 0 24 内含子4
Intron 4g.65A>G AA 14 36 60 110 61.2907 <0.0001 AG 32 24 25 81 GG 24 4 0 28 外显子6
Exon 6g.80T>A TT 32 32 0 64 77.6947 <0.0001 TA 24 32 45 101 AA 14 0 40 54 -
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