CAPN1基因对鸡肌肉嫩度性状的遗传效应及其表达规律研究
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摘要
本试验选取我国广东清远市的优质鸡品种-清远麻鸡和一个国外引进鸡品种-隐性白羽鸡作为研究对象,将CAPN1基因作为影响鸡肉嫩度性状的候选基因,采用PCR-SSCP和测序相结合的方法,在清远麻鸡和隐性白羽鸡群体中对CAPN1基因进行单核苷酸多态性(SNPs)扫描并分析其与肌肉嫩度等肉质性状之间的关系;同时利用SYBR Green I荧光定量PCR法检测了CAPN1基因在清远麻鸡和隐性白羽鸡胸肌和腓肠肌组织中的时空表达规律。试验结果如下:
1. CAPN1基因多态性检测
采用PCR-SSCP的方法对清远麻鸡和隐性白羽鸡CAPN1基因的编码区序列及3′调控区部分序列进行了SNPs检测。结果在3对引物的扩增序列中检测到了多态性,分别为外显子5中T2507C突变,C2546T突变,外显子6中G3535A突变以及3′调控区的G9950A突变,位于编码区的3个突变均没有引起相应氨基酸的改变,故均为沉默突变。统计了不同多态位点在不同品种中的基因型频率和基因频率,发现基因型分布在不同品种中存在差异。外显子5的多态位点在隐性白羽鸡中发现了A、B 2个等位基因和AA、AB、BB三种基因型, AA型为野生型,BB型在2546处存在C/T突变,其中B等位基因频率(0.752)明显高于A等位基因频率(0.248),在清远麻鸡中则发现了A、B、C 3个等位基因和AA、AB、BB、AC、BC、CC六种基因型,CC型个体同时存在T2507C突变和C2546T突变,其中C等位基因的频率为0.188;3′调控区的多态位点在两个鸡品种中均发现了G、A两个等位基因和GG、GA、AA三种基因型,在清远麻鸡群体中G等位基因的频率(0.675)高于A等位基因频率(0.325),在隐性白羽鸡群体中G等位基因的频率(0.484)则略低于A等位基因频率(0.516);外显子6在两个鸡品种中均发现了G、A两个等位基因和GG、GA、AA三种基因型,在清远麻鸡群体中G等位基因的频率(0.190)明显低于A等位基因频率(0.810),而在隐性白羽鸡中则完全相反,G等位基因的频率(0.840)明显高于A等位基因频率(0.160)。
2. CAPN1基因各SNPs位点与肉质性状的关系
不同品种、不同基因型的个体剪切力值最小二乘分析结果表明:在SNPC5位点上,存在显著的品种效应,在隐性白羽鸡中存在显著的基因型效应,AB型和BB型个体的剪切力值显著高于AA型(P<0.05),AB型个体剪切力值稍高于BB型个体,但两者之间的差异不显著;而清远麻鸡中基因型效应却不显著。在SNPC6位点上,存在显著的品种效应,在隐性白羽鸡中不存在显著的基因型效应;而在清远麻鸡中,GA型和AA型个体的剪切力值显著高于GG型(P<0.05)。在SNPC3的多态位点上,品种效应,基因型效应以及品种与基因型之间的互作效应均不显著。
不同品种、不同基因型的个体系水力值最小二乘分析结果表明:在SNPC5位点上,存在显著的品种效应,在隐性白羽鸡中存在显著的基因型效应,AB型的系水力显著高于BB型(P<0.05)。在SNPC6和SNPC3位点上均不存在显著的品种效应、基因型效应以及品种与基因型之间的互作效应。(注:SNPC3、SNPC5、SNPC6分别代表CAPN1基因3′调控区、外显子5、外显子6的SNP位点,下面同。)
3. CAPN1基因在鸡不同生长阶段表达规律研究
在清远麻鸡和隐性白羽鸡的不同生长阶段(0,2,4,6,8,10,12周龄)胸肌和腓肠肌组织中均检测到了CAPN1基因的mRNA。在胸肌中,清远麻鸡CAPN1基因表达水平高于隐性白羽鸡,但两个鸡品种各时期发育性变化规律趋于一致,且表达高峰均出现在6周龄;而在腓肠肌中清远麻鸡2.5周龄以后的表达水平低于隐性白羽鸡,2.5周龄以前略低于隐性白羽鸡。两个鸡品种表达的变化趋势显示,以8周龄为分界线,8周龄以前两者的表达水平呈现相反的变化趋势,8周龄以后变化趋于一致,且清远麻鸡的表达高峰是在2周龄,而隐性白羽鸡的表达高峰出现在6周龄。
同一鸡品种在不同组织的表达水平对比结果显示,两个鸡品种的对比结果类似,均为腓肠肌内的表达水平高于胸肌。从变化趋势来看,2周龄以前清远麻鸡在胸肌和腓肠肌内的变化趋势一致,2周龄以后的变化趋势则完全不一致,胸肌内的表达高峰期为6周龄,腓肠肌内为2周龄;隐性白羽鸡在胸肌和腓肠肌中的表达趋势为8周龄以前一致,8周龄以后不一致,表达高峰均出现在6周龄。
This experiment selects our country Guangdong Qingyuan's high quality chicken variety - Qingyuan partridge chicken and an overseas introduction chicken plants - Recessive White chicken to take the object of study, takes CAPN1 gene as candidate gene for chicken meat tenderness, PCR-SSCP and sequencing methods were used to investigate single nucleotide polymorphisms in CAPN1 gene of the Qingyuan partridge chicken and the Recessive White chicken,also,analyze the relationship between CAPN1 gene and the muscle tenderness; Meanwhile examined the CAPN1 gene' space-time expression rule using SYBR Green I fluorescence quota PCR in the Qingyuan partridge chicken and the Recessive White chicken pectoral muscle and gastrocnemius muscle. The test result is as follows:
High quality chicken breeds - Qingyuan partridge chicken (QY) from Qingyuan of Guangdong Province and an exotic breed - Recessive White chicken (RW) were taken in this research, and CAPN1 gene was taken as candidate gene for meat tenderness in chicken, single strand conformation (SSCP) analysis and sequencing reactions were performed to screen single nucleotide polymorphisms (SNPs) in Qingyuan partridge chicken and Recessive White chicken, and the correlation between results of SNPs and meat tenderness was analyzed; further, gene expression of CAPN1 gene in pectoral muscle and gastrocnemius muscle in Qingyuan partridge chicken and Recessive White chicken was investigated by SYBR Green I Real-time PCR technology to explore the expression characteristics, The main results were as follows:
1. Polymorphism of CAPN1 gene
Polymerase chain reaction - single strand conformation (PCR-SSCP) was performed to detect the SNPs of segment sequences in CAPN1 gene in Qingyuan partridge chicken and Recessive White chicken. The result shows three SNPs were detected : T2507C and C2546T from exon 5, G3535A from exon 6,and G9950A from 3'UTR. All the mutations in the coding regions were silence mutations; they didn’t cause the alteration of the corresponding amino acid. After Calculated the frequency of genotypes and genes and discovered some differences in the distribution of genotype frequencies among different breeds,A、B alleles and AA、AB、BB genotypes were detected at exon 5 in Recessive White chicken,and AA for wild, BB had an mutation of C2546T, and frequency of B allele (0.752) is higher than A (0.248) obviously, then A, B, C alleles and AA, AB, BB, AC, BC, and CC genotypes were detected in Qingyuan partridge chicken, CC genotype had two mutations of T2507C and C2546T, and the frequency of C allele is 0.188; G, A alleles and GG, GA, AA genotypes were detected at 3'UTR in two chicken breeds, and the frequency of G allele (0.675) was higher than A (0.325) in Qingyuan partridge chicken, but the frequency of G allele (0.484) was lower than A (0.516) slightly in Recessive White chicken; G, A alleles and GG, GA, AA genotypes were detected at exon 6 in two chicken breeds, and the frequency of G allele (0.190) was lower than A (0.810) obviously in Qingyuan partridge chicken, on the contrary, the frequency of G allele (0.840) was higher than A allele frequency(0.160) obviously in Recessive White chicken.
2. The relationship between SNPs of CAPN1 gene and the meat quality
The least square estimation between genotypes、breeds and the shear force indicated:On the SNPC5, it had significant breed effect,genotype effects were significant in Recessive White chicken, AB and BB genotype birds had significant higher shear force than AA genotype birds (P<0.05),AB genotype birds had slight higher shear force than BB genotype birds, but the difference of shear force between AB and BB genotype birds were not significant; Yet genotype effects were not significant in Qingyuan partridge chicken . On the SNPC6, it had significant breed effect,genotype effects were not significant in Recessive White chicken; But GA and AA genotype birds had significant higher shear force than GG genotype birds (P<0.05). On the SNPC3, The breed effect、genotype effect and the interaction effect between breed and genotype are not significant.
The least square estimation between genotypes、breeds and the water holding capacity indicated: On the SNPC5, it had significant breed effect,genotype effects were significant in Recessive White chicken, AB genotype birds had significant higher water holding capacity than BB genotype birds (P<0.05). On the SNPC3 and SNPC6, The breed effect、genotype effect and the interaction effect between breed and genotype are not significant.
(Note: SNPC3、SNPC5、SNPC6 stand for SNPs site of 3'UTR、exon 5、exon 6 of the CAPN1 gene, the following is by parity of reasoning. )
3.The expression research for CAPN1 gene on chicken 's different growth phase
CAPN1 gene mRNA were detected in pectoral muscle and gastrocnemius muscle in different growth phase (0,2,4,6,8,10,12 week’s age) of the Qingyuan partridge chicken and the Recessive White chicken. Various ages pectoral muscle expression patterns of Qingyuan partridge chicken and the Recessive White chicken was identical, and the peak of expression both appeared at 6 week’s age; But within the gastrocnemius muscle, boundary was taken at 8 week’s age, the expression patterns of two chicken breeds was identical 8 week’s age later, and opposite tendency existed of two chicken breeds at 0 - 8 week’s age, and the peak of Qingyuan partridge chicken expression in gastrocnemius muscle appeared at 2 week’s age, but the peak of Recessive White chicken expression appeared at 6 week’s age.
The contrast results of the expression patterns of different tissue in identical chicken breed indicated: the expression patterns of Qingyuan partridge chicken in pectoral muscle and gastrocnemius muscle was identical at 0 - 2 week’s age , and differences existed in two tissues 2 week’s age later, and the peak of Qingyuan partridge chicken expression in pectoral muscle appeared at 6 week’s age, but the peak of expression in gastrocnemius muscle appeared at 2 week’s age. Within the Recessive White chicken, the expression patterns in pectoral muscle and gastrocnemius muscle was identical at 0 - 8 week’s age , and differences existed in two tissues 8 week’s age later, the peak of expression both appeared at 6 week’s age.
1. CAPN1基因多态性检测
采用PCR-SSCP的方法对清远麻鸡和隐性白羽鸡CAPN1基因的编码区序列及3′调控区部分序列进行了SNPs检测。结果在3对引物的扩增序列中检测到了多态性,分别为外显子5中T2507C突变,C2546T突变,外显子6中G3535A突变以及3′调控区的G9950A突变,位于编码区的3个突变均没有引起相应氨基酸的改变,故均为沉默突变。统计了不同多态位点在不同品种中的基因型频率和基因频率,发现基因型分布在不同品种中存在差异。外显子5的多态位点在隐性白羽鸡中发现了A、B 2个等位基因和AA、AB、BB三种基因型, AA型为野生型,BB型在2546处存在C/T突变,其中B等位基因频率(0.752)明显高于A等位基因频率(0.248),在清远麻鸡中则发现了A、B、C 3个等位基因和AA、AB、BB、AC、BC、CC六种基因型,CC型个体同时存在T2507C突变和C2546T突变,其中C等位基因的频率为0.188;3′调控区的多态位点在两个鸡品种中均发现了G、A两个等位基因和GG、GA、AA三种基因型,在清远麻鸡群体中G等位基因的频率(0.675)高于A等位基因频率(0.325),在隐性白羽鸡群体中G等位基因的频率(0.484)则略低于A等位基因频率(0.516);外显子6在两个鸡品种中均发现了G、A两个等位基因和GG、GA、AA三种基因型,在清远麻鸡群体中G等位基因的频率(0.190)明显低于A等位基因频率(0.810),而在隐性白羽鸡中则完全相反,G等位基因的频率(0.840)明显高于A等位基因频率(0.160)。
2. CAPN1基因各SNPs位点与肉质性状的关系
不同品种、不同基因型的个体剪切力值最小二乘分析结果表明:在SNPC5位点上,存在显著的品种效应,在隐性白羽鸡中存在显著的基因型效应,AB型和BB型个体的剪切力值显著高于AA型(P<0.05),AB型个体剪切力值稍高于BB型个体,但两者之间的差异不显著;而清远麻鸡中基因型效应却不显著。在SNPC6位点上,存在显著的品种效应,在隐性白羽鸡中不存在显著的基因型效应;而在清远麻鸡中,GA型和AA型个体的剪切力值显著高于GG型(P<0.05)。在SNPC3的多态位点上,品种效应,基因型效应以及品种与基因型之间的互作效应均不显著。
不同品种、不同基因型的个体系水力值最小二乘分析结果表明:在SNPC5位点上,存在显著的品种效应,在隐性白羽鸡中存在显著的基因型效应,AB型的系水力显著高于BB型(P<0.05)。在SNPC6和SNPC3位点上均不存在显著的品种效应、基因型效应以及品种与基因型之间的互作效应。(注:SNPC3、SNPC5、SNPC6分别代表CAPN1基因3′调控区、外显子5、外显子6的SNP位点,下面同。)
3. CAPN1基因在鸡不同生长阶段表达规律研究
在清远麻鸡和隐性白羽鸡的不同生长阶段(0,2,4,6,8,10,12周龄)胸肌和腓肠肌组织中均检测到了CAPN1基因的mRNA。在胸肌中,清远麻鸡CAPN1基因表达水平高于隐性白羽鸡,但两个鸡品种各时期发育性变化规律趋于一致,且表达高峰均出现在6周龄;而在腓肠肌中清远麻鸡2.5周龄以后的表达水平低于隐性白羽鸡,2.5周龄以前略低于隐性白羽鸡。两个鸡品种表达的变化趋势显示,以8周龄为分界线,8周龄以前两者的表达水平呈现相反的变化趋势,8周龄以后变化趋于一致,且清远麻鸡的表达高峰是在2周龄,而隐性白羽鸡的表达高峰出现在6周龄。
同一鸡品种在不同组织的表达水平对比结果显示,两个鸡品种的对比结果类似,均为腓肠肌内的表达水平高于胸肌。从变化趋势来看,2周龄以前清远麻鸡在胸肌和腓肠肌内的变化趋势一致,2周龄以后的变化趋势则完全不一致,胸肌内的表达高峰期为6周龄,腓肠肌内为2周龄;隐性白羽鸡在胸肌和腓肠肌中的表达趋势为8周龄以前一致,8周龄以后不一致,表达高峰均出现在6周龄。
This experiment selects our country Guangdong Qingyuan's high quality chicken variety - Qingyuan partridge chicken and an overseas introduction chicken plants - Recessive White chicken to take the object of study, takes CAPN1 gene as candidate gene for chicken meat tenderness, PCR-SSCP and sequencing methods were used to investigate single nucleotide polymorphisms in CAPN1 gene of the Qingyuan partridge chicken and the Recessive White chicken,also,analyze the relationship between CAPN1 gene and the muscle tenderness; Meanwhile examined the CAPN1 gene' space-time expression rule using SYBR Green I fluorescence quota PCR in the Qingyuan partridge chicken and the Recessive White chicken pectoral muscle and gastrocnemius muscle. The test result is as follows:
High quality chicken breeds - Qingyuan partridge chicken (QY) from Qingyuan of Guangdong Province and an exotic breed - Recessive White chicken (RW) were taken in this research, and CAPN1 gene was taken as candidate gene for meat tenderness in chicken, single strand conformation (SSCP) analysis and sequencing reactions were performed to screen single nucleotide polymorphisms (SNPs) in Qingyuan partridge chicken and Recessive White chicken, and the correlation between results of SNPs and meat tenderness was analyzed; further, gene expression of CAPN1 gene in pectoral muscle and gastrocnemius muscle in Qingyuan partridge chicken and Recessive White chicken was investigated by SYBR Green I Real-time PCR technology to explore the expression characteristics, The main results were as follows:
1. Polymorphism of CAPN1 gene
Polymerase chain reaction - single strand conformation (PCR-SSCP) was performed to detect the SNPs of segment sequences in CAPN1 gene in Qingyuan partridge chicken and Recessive White chicken. The result shows three SNPs were detected : T2507C and C2546T from exon 5, G3535A from exon 6,and G9950A from 3'UTR. All the mutations in the coding regions were silence mutations; they didn’t cause the alteration of the corresponding amino acid. After Calculated the frequency of genotypes and genes and discovered some differences in the distribution of genotype frequencies among different breeds,A、B alleles and AA、AB、BB genotypes were detected at exon 5 in Recessive White chicken,and AA for wild, BB had an mutation of C2546T, and frequency of B allele (0.752) is higher than A (0.248) obviously, then A, B, C alleles and AA, AB, BB, AC, BC, and CC genotypes were detected in Qingyuan partridge chicken, CC genotype had two mutations of T2507C and C2546T, and the frequency of C allele is 0.188; G, A alleles and GG, GA, AA genotypes were detected at 3'UTR in two chicken breeds, and the frequency of G allele (0.675) was higher than A (0.325) in Qingyuan partridge chicken, but the frequency of G allele (0.484) was lower than A (0.516) slightly in Recessive White chicken; G, A alleles and GG, GA, AA genotypes were detected at exon 6 in two chicken breeds, and the frequency of G allele (0.190) was lower than A (0.810) obviously in Qingyuan partridge chicken, on the contrary, the frequency of G allele (0.840) was higher than A allele frequency(0.160) obviously in Recessive White chicken.
2. The relationship between SNPs of CAPN1 gene and the meat quality
The least square estimation between genotypes、breeds and the shear force indicated:On the SNPC5, it had significant breed effect,genotype effects were significant in Recessive White chicken, AB and BB genotype birds had significant higher shear force than AA genotype birds (P<0.05),AB genotype birds had slight higher shear force than BB genotype birds, but the difference of shear force between AB and BB genotype birds were not significant; Yet genotype effects were not significant in Qingyuan partridge chicken . On the SNPC6, it had significant breed effect,genotype effects were not significant in Recessive White chicken; But GA and AA genotype birds had significant higher shear force than GG genotype birds (P<0.05). On the SNPC3, The breed effect、genotype effect and the interaction effect between breed and genotype are not significant.
The least square estimation between genotypes、breeds and the water holding capacity indicated: On the SNPC5, it had significant breed effect,genotype effects were significant in Recessive White chicken, AB genotype birds had significant higher water holding capacity than BB genotype birds (P<0.05). On the SNPC3 and SNPC6, The breed effect、genotype effect and the interaction effect between breed and genotype are not significant.
(Note: SNPC3、SNPC5、SNPC6 stand for SNPs site of 3'UTR、exon 5、exon 6 of the CAPN1 gene, the following is by parity of reasoning. )
3.The expression research for CAPN1 gene on chicken 's different growth phase
CAPN1 gene mRNA were detected in pectoral muscle and gastrocnemius muscle in different growth phase (0,2,4,6,8,10,12 week’s age) of the Qingyuan partridge chicken and the Recessive White chicken. Various ages pectoral muscle expression patterns of Qingyuan partridge chicken and the Recessive White chicken was identical, and the peak of expression both appeared at 6 week’s age; But within the gastrocnemius muscle, boundary was taken at 8 week’s age, the expression patterns of two chicken breeds was identical 8 week’s age later, and opposite tendency existed of two chicken breeds at 0 - 8 week’s age, and the peak of Qingyuan partridge chicken expression in gastrocnemius muscle appeared at 2 week’s age, but the peak of Recessive White chicken expression appeared at 6 week’s age.
The contrast results of the expression patterns of different tissue in identical chicken breed indicated: the expression patterns of Qingyuan partridge chicken in pectoral muscle and gastrocnemius muscle was identical at 0 - 2 week’s age , and differences existed in two tissues 2 week’s age later, and the peak of Qingyuan partridge chicken expression in pectoral muscle appeared at 6 week’s age, but the peak of expression in gastrocnemius muscle appeared at 2 week’s age. Within the Recessive White chicken, the expression patterns in pectoral muscle and gastrocnemius muscle was identical at 0 - 8 week’s age , and differences existed in two tissues 8 week’s age later, the peak of expression both appeared at 6 week’s age.
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[27]赵瑞.利用PCR-SSCP分析鸡绿壳蛋基因的SNP标记[J].农业生物技术学报,2006,14 (5):673-676.
[28]王彦,苏毅,刘益平,等.鸡MC3R基因多态性及其与屠体和肉质性状的相关性[J].畜牧兽医学报, 2007, 38(10):1027-1031.
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[32]黄艳群,杜晓惠,邓学梅,等.鸡lmbr1基因单核苷酸多态与生长及屠体性状的关联性[J].中国科学C辑生命科学,2006,36(3):232-239.
[33]孙文浩,朱庆,将小松,等.鸡MYF6基因遗传多态性及其遗传效应研究[J].遗传,2008,30(1):71-76.
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[36]李长龙.猪ADD1基因的克隆及其与肉质性状关系的研究[D].黑龙江哈尔滨:东北农业大学, 2004.
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[39] Freeman W M, Vrana K E. Quantitative RT-PCR: pitfalls and potential[J]. Biotechniques, 1999, 26: 112-125.
[40] Marone M, Giannitelli C, Giannitelli C. Cyclin E and cdk2 alterations in ovarian cancer: amplification and overexpression[J]. International Journal of Cancer, 1997, 74: 390-395.
[41] Razin E K, Leslie B, Schrader J W. Connective tissue mast cell in contact with fibroblasts express IL-3 mRNA: Analysis of single cells by polymerase chain reaction[J]. J. Immunol, 1991, 146: 981-987.
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[46] Galbiati S,Smid M,Gambini D,et a1.Fetal DNA detection in maternal plasma throughout gestation[J].Hum Genet,2005,117(2-3):243-248.
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[48]李娅,韦平,金元昌,等.实时荧光PCR检测鸡GAPDH基因方法的建立[J].中国家禽,2008, 30(8):37-40.
[49]李馨,肖翠红,杨隽,等.鹅生长激素受体基因克隆及其个体发育性表达研究[J],中国畜牧杂志,2008,44(23):9-13.
[50]张颖,刘长军,秦运安,等.应用双重实时荧光定量PCR方法检测鸡马立克氏病血清1型病毒[J].中国预防兽医学报,2008, 29(1): 46-51.
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[52]李丽芳,贾球锋,张映,等.禽胰多肽对肉鸡肝脏和小肠组织中APPR mRNA表达量影响的研究[J].动物营养学报,2009, 21(1):107-112.
[53]苏胜彦,李齐发,刘振山,等.朗德鹅填饲后不同组织PPARγ基因mRNA表达量差异的初步研究[J].畜牧兽医学报, 2008, 39(7): 879-884.
[54]苏胜彦,李齐发,刘振山,等.朗德鹅肝脏和脂肪组织LXRα基因表达水平的比较[J].农业生物技术学报, 2008, 16(3): 421-425.
[55]文艳玲,谢芝勋,王莹,等. I群禽腺病毒SYBR Green I荧光PCR检测方法的建立[J],中国兽医科学,2008,38(09):753-756.
[56]褚晓红,徐宁迎,胡锦平,等.浙东白鹅催乳素基因表达特点[J].遗传, 2008, 30(8): 1021-1025.
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[61]杨秀芹,刘慧,郭丽娟,等.猪CAPN1基因部分外显子及3′UTR区的SNPs检测[J].遗传,2008,30(6):741-746.
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[63]刘安芳,朱庆,刘益平,等.钙蛋白酶基因在鸡不同组织和品种中的表达差异[J].遗传育种,2007,43(19):4-7.
[64]刘安芳,朱庆,刘益平,等.钙蛋白酶抑制蛋白(CAST)基因在鸡不同组织和品种中的表达差异[J].中国农业科学,2007,40(10):2331-2335.
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