鸡脂肪酸结合蛋白相关候选基因遗传效应及表达规律研究
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摘要
脂肪酸结合蛋白(Fatty acid binding protein,FABP)是机体脂肪代谢过程的重要调控因子之一,其最基本的功能是参与细胞内脂肪酸的摄取,协助将脂肪酸运至甘油三酯和磷脂的合成部位以及进行β-氧化的场所。本研究在如皋黄鸡、安卡鸡、文昌鸡三个鸡种中对鸡A-FABP、H-FABP、Ex-FABP基因外显子和部分内含子进行单核苷酸多态性(SNPs)扫描并分析其与胸肌肌内脂肪含量之间的关系;同时采用荧光定量PCR技术分析如皋黄鸡A-FABP和Ex-FABP基因的不同基因型的时空表达规律及聚合基因型在腹脂组织中的表达;主要研究结果如下:
1.在如皋黄鸡、安卡鸡、文昌鸡三个鸡种中对A-FABP、H-FABP、Ex-FABP基因外显子和部分内含子进行单核苷酸多态性检测,结果发现A-FABP基因位于外显子3的1763 A/G位点突变,将核苷酸序列翻译成对应的氨基酸序列后,发现突变引起Ser突变为Asn;H-FABP基因的2980 A/G位点突变;Ex-FABP基因的内含子1的2913 A/G位点突变、Ex-FABP基因的内含子3的3654 C/T、3699 C/T、3700 A/G突变、Ex-FABP基因外显子4的4223 G/A、4271 G/A和位于内含子4的4281 G/A突变,这两个突变均为沉默突变。
2.A-FABP基因外显子3及Ex-FABP基因外显子4的突变位点上的不同基因型对12周龄的如皋黄鸡的胸肌肌内脂肪含量进行关联分析。结果发现不同性别的胸肌的肌内脂肪含量差异不显著(P>0.05),但母鸡的IMF含量比公鸡的IMF含量高;A-FABP基因三种基因型中的AA、AB型个体的IMF含量显著地高于BB型个体(P<0.05),AA型IMF含量最高,为有利基因型;Ex-FABP基因三种基因型中的BB型个体的IMF含量显著地高于AA型、AB型,BB型为有利基因型,AA型、AB型间差异不显著;A-FABP基因和Ex-FABP基因聚合时,A-FABP基因中的有利基因型AA型和Ex-FABP基因中的有利基因型BB型聚合后的AABB型在合并后仍然是最佳聚合基因型。初步认为A-FABP及Ex-FABP基因为肌肉肌内脂肪含量的候选标记基因。
3.A-FABP基因的三种基因型在时间上表达趋势基本一致,在第4周龄表达水平最高,6、8周龄缓慢下降,10周上升,12周龄继续下降。A-FABP不同基因型在不同组织的表达水平存在显著差异。在腹脂和胸肌中,均以AA型表达水平最高,与BB型的差异极显著(P<0.01)。心脏和肝脏中以AB型的表达水平最高,与BB型存在极显著地差异(P<0.01)。进一步表明A-FABP基因(BB型)可作为肌内脂肪含量候选标记。
4.Ex-FABP基因在4周龄前表达水平较高,4周龄以后开始下降,10-12周龄后的表达水平有所回升,不同基因型表达规律基本一致。Ex-FABP各基因型在不同组织的表达量没有显著差异,但是在心脏中表达量较高。Ex-FABP不同基因型个体在相同组织中的表达水平不存在显著差异。
5.本试验进一步检测4、8、12周龄三个时段如皋黄鸡A-FABP和Ex-FABP两基因聚合基因型在腹脂组织中的表达规律。结果发现A-FABP基因聚合基因型在不同时期表达量呈下降趋势,Ex-FABP基因聚合基因型在不同时期表达量也呈下降趋势。就两基因的聚合基因型而言,AAAB型的表达水平最高,BBAB表达水平低,聚合基因型效应与肌内脂肪含量差异基本相一致,表明聚合基因型可作为肌内脂肪含量的候选标记而加以应用。
Fatty acid binding protein (FABP) is one of the important regulatory factors in the body's fat metabolism process. The basic function of FABP is involved in intracellular fatty acid intake, and to help fatty acids transport to the site of triglycerides and phospholipids synthesis as well as the place ofβ-oxidation.This study have scanned the single nucleotide polymorphisms (SNPs) in the Exons and part of intron of the A-FABP, H-FABP, Ex-FABP genes among Rugao chicken, Anka chicken, Wenchang Chicken, and analyzed their relationship with intramuscular fat content in breast; simultaneously detected the expression of different genotypes in the various organizations and the pyramiding genotype in abdominal fat tissue between A-FABP and Ex-FABP in Rugao chicken by quantitative Real-Time RT-PCR. The main results were as follows:
1.PCR-SSCP method and DNA sequencing approach were used to identifying the single nucleotide polymorphisms (SNPs) of A-FABP、H-FABP、Ex-FABP genes in exons and part of intron among Rugao Cao chicken,Anka chicken, Wenchang Chicken, and a 1763 A/G mutation in Exon3 of A-FABP gene were found, which caused the alteration of the corresponging amino acid from Ser to Asn.Other mutations, including a 2980 A/G mutation of H-FABP gene, a 2913 A/G mutation in intron 1 of EX-FABP gene, mutations of 3654 C/T,3699 C/T,3700 A/G in intron 3, 4223 G/A and 4271 G/A mutations in Exon 4 of EX-FABP gene, were all silence mutations.
2.Association analysis the SNPs with intramuscular fat content of breast muscle belonged to the different genotypes of A-FABP Exon 3 and Ex-FABP Exon 4 in 12 weeks Rugao chicken, showed that the intramuscular fat content of breast muscle was not significant between different sex,while the IMF content of hen was higher than that of cock. In three genotypes of A-FABP gene ,IMF content of the AA and AB genotype was significantly higher than the BB genotype (P <0.05), so the AA genotype was the dominant type with the highest IMF content. Likewise, in three genotypes of Ex-FABP gene, the IMF content of the BB genotype, which was the dominant genotype, was significantly higher than that of the AA type and AB type, and there was no significant difference between the AA and AB genotype. After merging the dominant genotype AA of A-FABP and the dominant genotype BB of Ex-FABP,we found the AABB was still the best genotype. In conclusion, it can be preliminary inferred that A-FABP gene and Ex-FABP gene were effect markers on IMF content.
3. The expression of A-FABP gene of different tissues was high in 4 weeks old, then decline in 6 weeks old, but rebound after 10 weeks old, and remains to decline in 12 weeks old. There was significant difference between the mRNA expressions of different A-FABP genotypes in various organizations. The Expression of AA genotype was the highest in abdominal fat and breast muscle,which was significantly different from that of BB type; Besides, the expression of the AB genotype is the highest in heart and liver, which was significant different from that of BB genotype.
4.The expression of Ex-FABP gene was high in early stage before 4 weeks old, then decline after 4 weeks old, but rebound after 10-12 weeks old. There was no difference in various organizations, but the highest was in hearts. The Ex-FABP mRNA expression levels analyzed by Q-PCR technique revealed that there was no significant difference between different genotypes in various organizations.
5.We had further inspected the Expression of the pyramiding genotypes between A-FABP and Ex-FABP in the abdominal fat tissue at 4, 8, and 12 weeks old, showing that the epression of pyramiding genotype of the A-FABP decreased at different times, while the expression of the Ex-FABP pyramiding genotype also decreased at different times. In the pyramiding genotypes of both genes, the expression level of AABB was the highest, BBAB was the lowest, that is consistent with the differences in intramuscular fat content of the pyramiding genotypes. These results indicated that pyramiding genotypes could be used as one of candidate markers on IMF content.
1.在如皋黄鸡、安卡鸡、文昌鸡三个鸡种中对A-FABP、H-FABP、Ex-FABP基因外显子和部分内含子进行单核苷酸多态性检测,结果发现A-FABP基因位于外显子3的1763 A/G位点突变,将核苷酸序列翻译成对应的氨基酸序列后,发现突变引起Ser突变为Asn;H-FABP基因的2980 A/G位点突变;Ex-FABP基因的内含子1的2913 A/G位点突变、Ex-FABP基因的内含子3的3654 C/T、3699 C/T、3700 A/G突变、Ex-FABP基因外显子4的4223 G/A、4271 G/A和位于内含子4的4281 G/A突变,这两个突变均为沉默突变。
2.A-FABP基因外显子3及Ex-FABP基因外显子4的突变位点上的不同基因型对12周龄的如皋黄鸡的胸肌肌内脂肪含量进行关联分析。结果发现不同性别的胸肌的肌内脂肪含量差异不显著(P>0.05),但母鸡的IMF含量比公鸡的IMF含量高;A-FABP基因三种基因型中的AA、AB型个体的IMF含量显著地高于BB型个体(P<0.05),AA型IMF含量最高,为有利基因型;Ex-FABP基因三种基因型中的BB型个体的IMF含量显著地高于AA型、AB型,BB型为有利基因型,AA型、AB型间差异不显著;A-FABP基因和Ex-FABP基因聚合时,A-FABP基因中的有利基因型AA型和Ex-FABP基因中的有利基因型BB型聚合后的AABB型在合并后仍然是最佳聚合基因型。初步认为A-FABP及Ex-FABP基因为肌肉肌内脂肪含量的候选标记基因。
3.A-FABP基因的三种基因型在时间上表达趋势基本一致,在第4周龄表达水平最高,6、8周龄缓慢下降,10周上升,12周龄继续下降。A-FABP不同基因型在不同组织的表达水平存在显著差异。在腹脂和胸肌中,均以AA型表达水平最高,与BB型的差异极显著(P<0.01)。心脏和肝脏中以AB型的表达水平最高,与BB型存在极显著地差异(P<0.01)。进一步表明A-FABP基因(BB型)可作为肌内脂肪含量候选标记。
4.Ex-FABP基因在4周龄前表达水平较高,4周龄以后开始下降,10-12周龄后的表达水平有所回升,不同基因型表达规律基本一致。Ex-FABP各基因型在不同组织的表达量没有显著差异,但是在心脏中表达量较高。Ex-FABP不同基因型个体在相同组织中的表达水平不存在显著差异。
5.本试验进一步检测4、8、12周龄三个时段如皋黄鸡A-FABP和Ex-FABP两基因聚合基因型在腹脂组织中的表达规律。结果发现A-FABP基因聚合基因型在不同时期表达量呈下降趋势,Ex-FABP基因聚合基因型在不同时期表达量也呈下降趋势。就两基因的聚合基因型而言,AAAB型的表达水平最高,BBAB表达水平低,聚合基因型效应与肌内脂肪含量差异基本相一致,表明聚合基因型可作为肌内脂肪含量的候选标记而加以应用。
Fatty acid binding protein (FABP) is one of the important regulatory factors in the body's fat metabolism process. The basic function of FABP is involved in intracellular fatty acid intake, and to help fatty acids transport to the site of triglycerides and phospholipids synthesis as well as the place ofβ-oxidation.This study have scanned the single nucleotide polymorphisms (SNPs) in the Exons and part of intron of the A-FABP, H-FABP, Ex-FABP genes among Rugao chicken, Anka chicken, Wenchang Chicken, and analyzed their relationship with intramuscular fat content in breast; simultaneously detected the expression of different genotypes in the various organizations and the pyramiding genotype in abdominal fat tissue between A-FABP and Ex-FABP in Rugao chicken by quantitative Real-Time RT-PCR. The main results were as follows:
1.PCR-SSCP method and DNA sequencing approach were used to identifying the single nucleotide polymorphisms (SNPs) of A-FABP、H-FABP、Ex-FABP genes in exons and part of intron among Rugao Cao chicken,Anka chicken, Wenchang Chicken, and a 1763 A/G mutation in Exon3 of A-FABP gene were found, which caused the alteration of the corresponging amino acid from Ser to Asn.Other mutations, including a 2980 A/G mutation of H-FABP gene, a 2913 A/G mutation in intron 1 of EX-FABP gene, mutations of 3654 C/T,3699 C/T,3700 A/G in intron 3, 4223 G/A and 4271 G/A mutations in Exon 4 of EX-FABP gene, were all silence mutations.
2.Association analysis the SNPs with intramuscular fat content of breast muscle belonged to the different genotypes of A-FABP Exon 3 and Ex-FABP Exon 4 in 12 weeks Rugao chicken, showed that the intramuscular fat content of breast muscle was not significant between different sex,while the IMF content of hen was higher than that of cock. In three genotypes of A-FABP gene ,IMF content of the AA and AB genotype was significantly higher than the BB genotype (P <0.05), so the AA genotype was the dominant type with the highest IMF content. Likewise, in three genotypes of Ex-FABP gene, the IMF content of the BB genotype, which was the dominant genotype, was significantly higher than that of the AA type and AB type, and there was no significant difference between the AA and AB genotype. After merging the dominant genotype AA of A-FABP and the dominant genotype BB of Ex-FABP,we found the AABB was still the best genotype. In conclusion, it can be preliminary inferred that A-FABP gene and Ex-FABP gene were effect markers on IMF content.
3. The expression of A-FABP gene of different tissues was high in 4 weeks old, then decline in 6 weeks old, but rebound after 10 weeks old, and remains to decline in 12 weeks old. There was significant difference between the mRNA expressions of different A-FABP genotypes in various organizations. The Expression of AA genotype was the highest in abdominal fat and breast muscle,which was significantly different from that of BB type; Besides, the expression of the AB genotype is the highest in heart and liver, which was significant different from that of BB genotype.
4.The expression of Ex-FABP gene was high in early stage before 4 weeks old, then decline after 4 weeks old, but rebound after 10-12 weeks old. There was no difference in various organizations, but the highest was in hearts. The Ex-FABP mRNA expression levels analyzed by Q-PCR technique revealed that there was no significant difference between different genotypes in various organizations.
5.We had further inspected the Expression of the pyramiding genotypes between A-FABP and Ex-FABP in the abdominal fat tissue at 4, 8, and 12 weeks old, showing that the epression of pyramiding genotype of the A-FABP decreased at different times, while the expression of the Ex-FABP pyramiding genotype also decreased at different times. In the pyramiding genotypes of both genes, the expression level of AABB was the highest, BBAB was the lowest, that is consistent with the differences in intramuscular fat content of the pyramiding genotypes. These results indicated that pyramiding genotypes could be used as one of candidate markers on IMF content.
引文
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