黄牛NPY和HCRTR1基因的克隆表达及其遗传多样性研究
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摘要
下丘脑是调节能量稳态、食欲和生殖的重要中枢,摄食行为是通过位于下丘脑内的神经环路调控的,其内存在着复杂的食欲调节网络。神经肽Y(NPY)是中枢神经系统中一种非常重要的参与食欲调控的肽。其神经元在脑内分布广泛,在下丘脑内主要分布在弓状核区域,并形成相互投射的神经环路。
本研究采用PCR-SSCP及测序方法研究了四个品种黄牛群体共338个个体(其中秦川牛68头、南阳牛100头、郏县红牛130头、晋南牛40头)HCRTR1基因和NPY基因的单核苷酸多态性,分析了基因多态位点与牛部分经济性状的相关性。此外,对秦川牛NPY和HCRTR1基因进行了克隆、表达研究,得到如下结果:
1 HCRTR1基因在我国4个牛群体中的遗传多样性
本研究采用PCR-SSCP和测序方法首次分析了HCRTR1基因编码区全长在我国4个牛群体中的遗传多态性。在该区域共检测到11个SNP位点,其中4个SNP位点的碱基突变导致了氨基酸的改变,在322 bp处G>A的突变造成缬氨酸变成亮氨酸,481 bp处T>A突变造成色氨酸变成精氨酸,631 bp处C>T突变造成精氨酸变成色氨酸,736 bp处C>T突变造成精氨酸变成色氨酸。其余8处的碱基替换在氨基酸水平上均为同义突变。本研究对牛HCRTR1基因11个SNP位点所在4个牛群体进行Hardy-Weinberg平衡检验,结果发现只有部分SNP位点处于平衡状态。
2 HCRTR1基因多态位点与牛生长发育性状的关系
利用最小二乘拟合线性模型,对HCRTR1基因多态位点与牛部分经济性状进行显著性检验,结果表明,HCRTR1基因第2外显子位点不同基因型与秦川牛、南阳牛体重和体尺性状间最小二乘均值差异均不显著。对于郏县红牛而言,B、C和D型牛体斜长和管围显著长于A型牛(P<0.05);对于胸围和尻长,D型牛显著大于A型(P<0.05);在郏县红牛1～3岁和4～8岁时,B和D型牛的体高和体重显著高于A型牛(P<0.05)。相同时间内,D型牛体格的绝对生长发育最快,而A型牛最慢,主要表现在体高、体斜长、胸围、管围、尻长和体重性状上。384、420和423 bp处SNP位点多态信息含量为0.2778,郏县红牛的选育过程中适合作为分子标记用于辅助选择。
3 NPY基因在我国4个牛群体中遗传多样性
本研究利用PCR-SSCP与测序方法首次分析了NPY基因编码区全长及编码区侧翼序列在我国4个牛群体中的遗传多态性。检测到5个SNP位点,其中位于NPY基因第2外显子29 bp处C>A突变引起了氨基酸的改变,由苏氨酸突变为天冬酰氨。第1外显子的78 bp处C>G突变在氨基酸水平上为同义突变,另外3处的突变发生在非编码区。NPY基因5个SNP位点所在4个牛群体Hardy-Weinberg平衡检验结果表明,大部分SNP位点处于非平衡状态。多态信息含量分析结果表明,除秦川牛NPY基因3’端71 bp和南阳牛第2内含子1 725 bp为低度多态座位外,其它位点均为中度多态,适合作为分子标记。
4 NPY基因多态性与牛生长发育性状的关系
利用最小二乘拟合线性模型,对NPY基因多态位点不同基因型与牛部分经济性状进行显著性检验,结果表明,在NPY基因第3外显子及侧翼序列位点检测到4种基因型,在南阳牛6、12和18月龄时,A型牛体斜长、胸围均显著大于B型牛(P<0.05),发育到24月龄时,A型牛这两项体尺指标仍然大于B型牛,尽管没有达到显著水平。NPY基因3’端71 bp处的SNP位点多态信息含量为0.3457,适合作为选育体格较大的南阳牛一个分子标记。
5秦川牛NPY和HCRTR1基因的克隆、表达
本试验利用Overlap-PCR技术在国内首次扩增获得秦川牛NPY和HCRTR1基因全长CDS序列。NPY基因编码区全长为294 bp,与GenBank公布序列的同源性为100%。HCRTR1基因CDS序列全长为1 278 bp,编码425个氨基酸,与GenBank中海福特牛该基因的序列比对发现存在4处突变,分别为237 bp:G>C;273 bp:T>C;276 bp:C>T和480 bp:T>C。这4处突变均为同义突变。
将NPY和HCRTR1两个基因分别克隆至pET32a+原核表达载体,构建重组质粒,转化大肠杆菌BL21(DE3)后诱导融合蛋白表达。经Western-blot检测证实了NPY蛋白的表达。电泳检测分析发现NPY基因在大肠杆菌中高效表达,表达蛋白以可溶性蛋白和包涵体两种形式存在。该融合蛋白的分子量约为30 kD,与预测的NPY融合蛋白大小相吻合。而HCRTR1基因在大肠杆菌中没有表达。
利用his trap亲和层析柱纯化NPY融合蛋白后,用纯化的蛋白免疫家兔获得抗血清,ELISA检测表明该血清具有很高的特异性和效价,表明NPY融合蛋白具有很好的抗体免疫原性。
以获得的抗血清作为一抗,免疫组织化学分析结果表明,牛NPY蛋白在下丘脑弓状核神经细胞的细胞膜和细胞质表达。
The hypothalamus being the central feeding and energy equation organ mediates regulation of dietary intake via synthesis of various orexigenic and anorectic neuropeptides. Neuropeptide Y was the key factor of nervous central in appetite regulation and distributed mainly in arcurate nucleus. HCRTR1 was the peripheral peptide and involved in appetite regulation. By now, fewer papers about bovine NPY and HCRTR1 gene were reported.
PCR-SSCP and sequencing assay technologies were applied to detect SNPs of NPY and HCRTR1 gene in four Chiense cattle breeds (Nanyang cattle, 100; Jiaxian cattle, 130; Qinchuan cattle, 68; and Jinnan cattle, 40). The associations between these polymorphisms and some cattle economic traits were analyzed using general linear model (GLM) with software SPSS 12.0. Cloning and expression of NPY and HCRTR1gene in E.coli were also studied in Qinchuan cattle. This work would lay a foundation for cattle breeding. Results were obtained as follows:
1 Polymorphisms of HCRTR1 gene in four breeds of Chinese cattle
338 cattle from four Chinese four cattle breeds were used for SNPs discovery in the complete coding region of HCRTR1 gene using PCR-SSCP and sequencing methods. Eleven SNPs were detected in the complete coding region of HCRTR1 gene. The variations at 322, 481, 631 and 736 bp caused amino acid replacements and variations at the other 7 SNPs were synonymous. Not all of the loci were at Hardy-Weinberg equilibration(P>0.05).
2 Effects of HCRTR1 gene polymorphisms on traits in cattle
No significant association was found between genotypes of exon 2 and the growth traits of Qinchuan and Nanyang cattle(P>0.05). For Jiaxian cattle, the value of body length and cannon circumference in B, C and D genotype was larger than that in A genotype (P<0.05). The value of heart girth and rump length in D genotype was larger than that in A genotype (P<0.05). The value of withers height and body weight in B and D genotype was larger than that in A genotype at the age of 1-8 years old (P<0.05). From above, cattle in D genotype grew faster than those in A genotype. SNPs at 384, 420 and 423bp can be fit for genetic markers in Jiaxian cattle breeding.
3 Polymorphisms of NPY gene in four breeds of Chinese cattle
338 cattle from four Chinese cattle breeds were used for SNPs discovery in the region of exon 1, 2, 3 and their bandary sequence of NPY gene using PCR-SSCP and sequencing methods. Five SNPs were discovered. The variations at the 29th bp of exon 2 caused amino acid replacement: from threonine to asparagines. Most of loci were not at Hardy-Weinberg equilibration(P <0.05)in these four populations. PIC of the locus at 71th bp in the 3’end of NPY gene in Qinchuan cattle and the locus at 1725th bp in intron 2 of NPY gene in Nanyang cattle were low polymorphic and the other loci were moderate polymorphic.
4 Effects of polymorphisms of NPY gene on traits in cattle
In locus of exon3 region, the value of body length and heart girth of Nanyang cattle in A genotype was longer than that in B genotype at the age of six, twelve and eighteen months age (P <0.05). At the age of 24 months the value of this two traits in A genotype was still larger than that in B genotype though the significant level can’t be attained to. The SNP at 71th bp of 3’end of NPY gene can be used as a genetic marker for breeding.
5 Cloning and expression of NPY and HCRTR1 gene in Qinchuan cattle
The coding sequence of NPY and HCRTR1 gene of Qinchuan cattle was amplified by overlap-PCR and confirmed with sequencing assay. The recombinant expressive plasmid pET32a+-NPY and pET32a+-HCRTR1 were transduced into E.coli strain BL21(DE3) and were induced to express by IPTG. In result, the recombinant plasmid pET32a+-NPY were over-expressed in E.coli as solubility and inclusion body with molecular weight 30 kD or so. But the recombinant plasmid pET32a+-HCRTR1 were not expressed in E.coli. We immunized rabbit using the purified fusion protein. The result of animal experiment showed that the fusion protein could stimulate animals to produce special and sensitive antibody. Using the obtained antiserum as a testing reagent combining with immunity histochemistry technology, we identified NPY as a coding protein which expressed specially in cellular cytoplast and cellular membrane of neuron in hypothalamic arcuate nucleus of cattle.
本研究采用PCR-SSCP及测序方法研究了四个品种黄牛群体共338个个体(其中秦川牛68头、南阳牛100头、郏县红牛130头、晋南牛40头)HCRTR1基因和NPY基因的单核苷酸多态性,分析了基因多态位点与牛部分经济性状的相关性。此外,对秦川牛NPY和HCRTR1基因进行了克隆、表达研究,得到如下结果:
1 HCRTR1基因在我国4个牛群体中的遗传多样性
本研究采用PCR-SSCP和测序方法首次分析了HCRTR1基因编码区全长在我国4个牛群体中的遗传多态性。在该区域共检测到11个SNP位点,其中4个SNP位点的碱基突变导致了氨基酸的改变,在322 bp处G>A的突变造成缬氨酸变成亮氨酸,481 bp处T>A突变造成色氨酸变成精氨酸,631 bp处C>T突变造成精氨酸变成色氨酸,736 bp处C>T突变造成精氨酸变成色氨酸。其余8处的碱基替换在氨基酸水平上均为同义突变。本研究对牛HCRTR1基因11个SNP位点所在4个牛群体进行Hardy-Weinberg平衡检验,结果发现只有部分SNP位点处于平衡状态。
2 HCRTR1基因多态位点与牛生长发育性状的关系
利用最小二乘拟合线性模型,对HCRTR1基因多态位点与牛部分经济性状进行显著性检验,结果表明,HCRTR1基因第2外显子位点不同基因型与秦川牛、南阳牛体重和体尺性状间最小二乘均值差异均不显著。对于郏县红牛而言,B、C和D型牛体斜长和管围显著长于A型牛(P<0.05);对于胸围和尻长,D型牛显著大于A型(P<0.05);在郏县红牛1～3岁和4～8岁时,B和D型牛的体高和体重显著高于A型牛(P<0.05)。相同时间内,D型牛体格的绝对生长发育最快,而A型牛最慢,主要表现在体高、体斜长、胸围、管围、尻长和体重性状上。384、420和423 bp处SNP位点多态信息含量为0.2778,郏县红牛的选育过程中适合作为分子标记用于辅助选择。
3 NPY基因在我国4个牛群体中遗传多样性
本研究利用PCR-SSCP与测序方法首次分析了NPY基因编码区全长及编码区侧翼序列在我国4个牛群体中的遗传多态性。检测到5个SNP位点,其中位于NPY基因第2外显子29 bp处C>A突变引起了氨基酸的改变,由苏氨酸突变为天冬酰氨。第1外显子的78 bp处C>G突变在氨基酸水平上为同义突变,另外3处的突变发生在非编码区。NPY基因5个SNP位点所在4个牛群体Hardy-Weinberg平衡检验结果表明,大部分SNP位点处于非平衡状态。多态信息含量分析结果表明,除秦川牛NPY基因3’端71 bp和南阳牛第2内含子1 725 bp为低度多态座位外,其它位点均为中度多态,适合作为分子标记。
4 NPY基因多态性与牛生长发育性状的关系
利用最小二乘拟合线性模型,对NPY基因多态位点不同基因型与牛部分经济性状进行显著性检验,结果表明,在NPY基因第3外显子及侧翼序列位点检测到4种基因型,在南阳牛6、12和18月龄时,A型牛体斜长、胸围均显著大于B型牛(P<0.05),发育到24月龄时,A型牛这两项体尺指标仍然大于B型牛,尽管没有达到显著水平。NPY基因3’端71 bp处的SNP位点多态信息含量为0.3457,适合作为选育体格较大的南阳牛一个分子标记。
5秦川牛NPY和HCRTR1基因的克隆、表达
本试验利用Overlap-PCR技术在国内首次扩增获得秦川牛NPY和HCRTR1基因全长CDS序列。NPY基因编码区全长为294 bp,与GenBank公布序列的同源性为100%。HCRTR1基因CDS序列全长为1 278 bp,编码425个氨基酸,与GenBank中海福特牛该基因的序列比对发现存在4处突变,分别为237 bp:G>C;273 bp:T>C;276 bp:C>T和480 bp:T>C。这4处突变均为同义突变。
将NPY和HCRTR1两个基因分别克隆至pET32a+原核表达载体,构建重组质粒,转化大肠杆菌BL21(DE3)后诱导融合蛋白表达。经Western-blot检测证实了NPY蛋白的表达。电泳检测分析发现NPY基因在大肠杆菌中高效表达,表达蛋白以可溶性蛋白和包涵体两种形式存在。该融合蛋白的分子量约为30 kD,与预测的NPY融合蛋白大小相吻合。而HCRTR1基因在大肠杆菌中没有表达。
利用his trap亲和层析柱纯化NPY融合蛋白后,用纯化的蛋白免疫家兔获得抗血清,ELISA检测表明该血清具有很高的特异性和效价,表明NPY融合蛋白具有很好的抗体免疫原性。
以获得的抗血清作为一抗,免疫组织化学分析结果表明,牛NPY蛋白在下丘脑弓状核神经细胞的细胞膜和细胞质表达。
The hypothalamus being the central feeding and energy equation organ mediates regulation of dietary intake via synthesis of various orexigenic and anorectic neuropeptides. Neuropeptide Y was the key factor of nervous central in appetite regulation and distributed mainly in arcurate nucleus. HCRTR1 was the peripheral peptide and involved in appetite regulation. By now, fewer papers about bovine NPY and HCRTR1 gene were reported.
PCR-SSCP and sequencing assay technologies were applied to detect SNPs of NPY and HCRTR1 gene in four Chiense cattle breeds (Nanyang cattle, 100; Jiaxian cattle, 130; Qinchuan cattle, 68; and Jinnan cattle, 40). The associations between these polymorphisms and some cattle economic traits were analyzed using general linear model (GLM) with software SPSS 12.0. Cloning and expression of NPY and HCRTR1gene in E.coli were also studied in Qinchuan cattle. This work would lay a foundation for cattle breeding. Results were obtained as follows:
1 Polymorphisms of HCRTR1 gene in four breeds of Chinese cattle
338 cattle from four Chinese four cattle breeds were used for SNPs discovery in the complete coding region of HCRTR1 gene using PCR-SSCP and sequencing methods. Eleven SNPs were detected in the complete coding region of HCRTR1 gene. The variations at 322, 481, 631 and 736 bp caused amino acid replacements and variations at the other 7 SNPs were synonymous. Not all of the loci were at Hardy-Weinberg equilibration(P>0.05).
2 Effects of HCRTR1 gene polymorphisms on traits in cattle
No significant association was found between genotypes of exon 2 and the growth traits of Qinchuan and Nanyang cattle(P>0.05). For Jiaxian cattle, the value of body length and cannon circumference in B, C and D genotype was larger than that in A genotype (P<0.05). The value of heart girth and rump length in D genotype was larger than that in A genotype (P<0.05). The value of withers height and body weight in B and D genotype was larger than that in A genotype at the age of 1-8 years old (P<0.05). From above, cattle in D genotype grew faster than those in A genotype. SNPs at 384, 420 and 423bp can be fit for genetic markers in Jiaxian cattle breeding.
3 Polymorphisms of NPY gene in four breeds of Chinese cattle
338 cattle from four Chinese cattle breeds were used for SNPs discovery in the region of exon 1, 2, 3 and their bandary sequence of NPY gene using PCR-SSCP and sequencing methods. Five SNPs were discovered. The variations at the 29th bp of exon 2 caused amino acid replacement: from threonine to asparagines. Most of loci were not at Hardy-Weinberg equilibration(P <0.05)in these four populations. PIC of the locus at 71th bp in the 3’end of NPY gene in Qinchuan cattle and the locus at 1725th bp in intron 2 of NPY gene in Nanyang cattle were low polymorphic and the other loci were moderate polymorphic.
4 Effects of polymorphisms of NPY gene on traits in cattle
In locus of exon3 region, the value of body length and heart girth of Nanyang cattle in A genotype was longer than that in B genotype at the age of six, twelve and eighteen months age (P <0.05). At the age of 24 months the value of this two traits in A genotype was still larger than that in B genotype though the significant level can’t be attained to. The SNP at 71th bp of 3’end of NPY gene can be used as a genetic marker for breeding.
5 Cloning and expression of NPY and HCRTR1 gene in Qinchuan cattle
The coding sequence of NPY and HCRTR1 gene of Qinchuan cattle was amplified by overlap-PCR and confirmed with sequencing assay. The recombinant expressive plasmid pET32a+-NPY and pET32a+-HCRTR1 were transduced into E.coli strain BL21(DE3) and were induced to express by IPTG. In result, the recombinant plasmid pET32a+-NPY were over-expressed in E.coli as solubility and inclusion body with molecular weight 30 kD or so. But the recombinant plasmid pET32a+-HCRTR1 were not expressed in E.coli. We immunized rabbit using the purified fusion protein. The result of animal experiment showed that the fusion protein could stimulate animals to produce special and sensitive antibody. Using the obtained antiserum as a testing reagent combining with immunity histochemistry technology, we identified NPY as a coding protein which expressed specially in cellular cytoplast and cellular membrane of neuron in hypothalamic arcuate nucleus of cattle.
引文
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