乌苏里貉FSHβ和FSHR基因多态性与繁殖性状的相关性研究
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摘要
乌苏里貉(Nyctereutes Ussurienusis Maxschie)是一种珍贵的毛皮动物,它以独特的针毛而出名,近年来随着毛皮市场的复苏,给毛皮动物带来良好的发展机遇和前途,因此,提高乌苏里貉的产仔数和增加乌苏里貉的存栏数可以有效的提高乌苏里貉的经养殖益。由于传统的育种技术带给乌苏里貉遗传表现型之间的差异,在种群内乌苏里貉繁殖性状差异很大,给经济动物饲养带来了很大的经济损失。因此运用现代分子生物学的研究提高产仔数,对乌苏里貉育种具有重要意义。
本研究以候选基因方法选取促卵泡素β亚基(FSHβ)及其受体(FSHR)基因作为候选基因。在家畜的研究中,FSHβ及其受体已被确定为繁殖性状的主效基因之一。根据已知基因的基础研究,以哈尔滨江北乌苏里貉养殖场提供的乌苏里貉为实验群体,采用单链构象多态性(PCR-SSCP)技术和DNA克隆测序的方法检测FSHβ基因和FSHR基因中的单核苷酸多态性(SNPs),分析基因多态位点与繁殖性状之间的相关性。主要研究结果如下：
(1)首次克隆测序了乌苏里貉FSHβ基因,包括第1-3外显子,第1和2内含子,以及部分5′上游调控序列,序列全长为3246 bp,已经发布到Genbank,登录号为HQ385979。
(2)利用PCR-SSCP技术,检测了乌苏里貉FSHβ基因的2个多态位点,分别是位于5′端调控区的G192A和外显子3的A2393G。并计算了各基因多态位点的相应基因型频率和等位基因频率。应用SNPs与繁殖性状的相关性分析,结果为2393G突变产生的CC、CD和DD三种基因型对乌苏里貉产仔数和产活仔数有一定影响(P<0.2),其中DD基因型个体对乌苏里貉产仔数的影响显著高于CC基因型。而G192A突变产生的AA、BB和AB基因型个体与产仔数和产活仔数之间没有显著性差异。
(3)克隆测序了乌苏里貉FSHR基因的外显子10和FSHR的5端序列。外显子10序列长1398 bp,Genbank登录号为HQ 385978；FSHR的5'端序列包括调控区序列和第1外显子序列,长度为757 bp, Genbank登录号为HQ 385977.
(4)将野生与家养乌苏里貉的FSHR基因5′端序列和外显子10进行比较,在野生乌苏里貉FSHR基因中有多处突变,包括：46～49位TTAG插入,452-453位AC插入、448-451位ACAC缺失,以及序列中9个SNPs位点,而在家养貉中只有1个SNPs位点。乌苏里貉的外显子10保守性很强,在野生貉中仅找到3个多态位点。
(5)利用PCR-SSCP检测到FSHR基因中2个多态性位点,5′端上游调控区的T133A和第1外显子的T623C,计算了这两个多态性位点相应的基因型频率和等位基因频率。通过相关性分析显示,两个SNPs位点对乌苏里貉的产仔数和产活仔数的影响差异都不显著。
Wusuli Raccoon Dog is a valuable animal whose skin is covered with fur. It became famous for tenacity of fur and unique guard hair. In recent years, it could provide good opportunities of development for fur-bearing animals with the market of fur recoveried. Therefore, the economic benefits of wusuli raccoon dog could be effectively improved by increasing the litter size and account of livestock on hand. The differences of genetic phenotypes between wusuli raccoon dogs were brought by traditional breeding techniques, and the differences of reproductive traits of populations were the largest, which brought great economic losses to feed economic animal. Therefore, it's important to improve litter size with the modern molecular biology techniques for the wusuli raccoon dog breeding.
Follicle-stimulating hormone beta subunit (FSHβ)and its receptor (FSHR) gene were viewed as the candidate gene. In the studies in livestock, FSHβand its receptors have been identified as major genes of reproductive traits.
In our study, the experimental groups were the wusuli raccoon dogs feeding in the farms of the wusuli raccoon. dogs in Jiangbei of Harbin. The single nucleotide polymorphisms (SNPs) of gene FSHβand FSHR gene were detected with the techniques of single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods. The correlation between gene polymorphism and reproductive traits analysis was analyzed. The main results are as follows: 1. FSHβgene was cloned and sequenced in the Wusuli raccoon dog. It's full-length was 3246bp. This sequences included the first 1 to 3 exons, intron 1 and 2, and the partial 5'upstream regulatory sequence of FSHβ. It has been released to the Genbank, and it's accession number is HQ 385979.
2. Two polymorphic sites of FSHβgene were detected in the Wusuli raccoon dog with PCR-SSCP technique. They are respectively located in G192A of 5'regulatory region and A2393G in exon 3. The corresponding frequencies of genotype and allele of the genetic polymorphisms were calculated. The correlative analysis of SNPs and the reproductive traits showed that the CC,CD and DD genotypes resulting from the A2393G mutation has some influence on litter size of the Wusuli raccoon dog (P<0.2). The DD genotype has a greater effect on the litter size than CC genotype in the Wusuli raccoon dog. There were not significant differences among litter size of the AA, BB and AB genotype caused by the G192A mutation.
3. Exon 10 and the 5'sequence of FSHR genes were cloned and sequenced in the Wusuli raccoon dog. The length of exon 10 sequence was 1398bp and it has been released to the Genbank. It's accession number was HQ 385979. The length of 5'sequence of FSHR was 757bp which included regulatory sequences and exon 1 sequence. It's Genbank accession number was HQ385977.
4. The comparison of 5'sequence and exon 10 of FSHR gene between wild and domesticated Wusuli raccoon dog showed that there were multiple mutations in the FSHR gene in the wild Wusuli raccoon dog. These mutations included 46 to 49 TTAG insertion sites,452～453AC insert,448～451 missing ACAC, and 9 SNPs in the sequence sites, while only one SNPs in the domesticated raccoon dogs. Exon 10 was highly conserved, only 3 polymorphisms was found in exon10 of FSHR in wild Wusuli raccoon dog.
5. Two polymorphic sites of FSHR gene were detected in the Wusuli raccoon with PCR-SSCP technique. They are located in the T133A of 5'regulatory region the and T623C of exon 1.
The corresponding frequencies of genotype and allele of these two polymorphisms were calculated. The correlation analysis showed that there were not significant differences between the litter size of these two SNPs in the Wusuli raccoon dog.
本研究以候选基因方法选取促卵泡素β亚基(FSHβ)及其受体(FSHR)基因作为候选基因。在家畜的研究中,FSHβ及其受体已被确定为繁殖性状的主效基因之一。根据已知基因的基础研究,以哈尔滨江北乌苏里貉养殖场提供的乌苏里貉为实验群体,采用单链构象多态性(PCR-SSCP)技术和DNA克隆测序的方法检测FSHβ基因和FSHR基因中的单核苷酸多态性(SNPs),分析基因多态位点与繁殖性状之间的相关性。主要研究结果如下：
(1)首次克隆测序了乌苏里貉FSHβ基因,包括第1-3外显子,第1和2内含子,以及部分5′上游调控序列,序列全长为3246 bp,已经发布到Genbank,登录号为HQ385979。
(2)利用PCR-SSCP技术,检测了乌苏里貉FSHβ基因的2个多态位点,分别是位于5′端调控区的G192A和外显子3的A2393G。并计算了各基因多态位点的相应基因型频率和等位基因频率。应用SNPs与繁殖性状的相关性分析,结果为2393G突变产生的CC、CD和DD三种基因型对乌苏里貉产仔数和产活仔数有一定影响(P<0.2),其中DD基因型个体对乌苏里貉产仔数的影响显著高于CC基因型。而G192A突变产生的AA、BB和AB基因型个体与产仔数和产活仔数之间没有显著性差异。
(3)克隆测序了乌苏里貉FSHR基因的外显子10和FSHR的5端序列。外显子10序列长1398 bp,Genbank登录号为HQ 385978；FSHR的5'端序列包括调控区序列和第1外显子序列,长度为757 bp, Genbank登录号为HQ 385977.
(4)将野生与家养乌苏里貉的FSHR基因5′端序列和外显子10进行比较,在野生乌苏里貉FSHR基因中有多处突变,包括：46～49位TTAG插入,452-453位AC插入、448-451位ACAC缺失,以及序列中9个SNPs位点,而在家养貉中只有1个SNPs位点。乌苏里貉的外显子10保守性很强,在野生貉中仅找到3个多态位点。
(5)利用PCR-SSCP检测到FSHR基因中2个多态性位点,5′端上游调控区的T133A和第1外显子的T623C,计算了这两个多态性位点相应的基因型频率和等位基因频率。通过相关性分析显示,两个SNPs位点对乌苏里貉的产仔数和产活仔数的影响差异都不显著。
Wusuli Raccoon Dog is a valuable animal whose skin is covered with fur. It became famous for tenacity of fur and unique guard hair. In recent years, it could provide good opportunities of development for fur-bearing animals with the market of fur recoveried. Therefore, the economic benefits of wusuli raccoon dog could be effectively improved by increasing the litter size and account of livestock on hand. The differences of genetic phenotypes between wusuli raccoon dogs were brought by traditional breeding techniques, and the differences of reproductive traits of populations were the largest, which brought great economic losses to feed economic animal. Therefore, it's important to improve litter size with the modern molecular biology techniques for the wusuli raccoon dog breeding.
Follicle-stimulating hormone beta subunit (FSHβ)and its receptor (FSHR) gene were viewed as the candidate gene. In the studies in livestock, FSHβand its receptors have been identified as major genes of reproductive traits.
In our study, the experimental groups were the wusuli raccoon dogs feeding in the farms of the wusuli raccoon. dogs in Jiangbei of Harbin. The single nucleotide polymorphisms (SNPs) of gene FSHβand FSHR gene were detected with the techniques of single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods. The correlation between gene polymorphism and reproductive traits analysis was analyzed. The main results are as follows: 1. FSHβgene was cloned and sequenced in the Wusuli raccoon dog. It's full-length was 3246bp. This sequences included the first 1 to 3 exons, intron 1 and 2, and the partial 5'upstream regulatory sequence of FSHβ. It has been released to the Genbank, and it's accession number is HQ 385979.
2. Two polymorphic sites of FSHβgene were detected in the Wusuli raccoon dog with PCR-SSCP technique. They are respectively located in G192A of 5'regulatory region and A2393G in exon 3. The corresponding frequencies of genotype and allele of the genetic polymorphisms were calculated. The correlative analysis of SNPs and the reproductive traits showed that the CC,CD and DD genotypes resulting from the A2393G mutation has some influence on litter size of the Wusuli raccoon dog (P<0.2). The DD genotype has a greater effect on the litter size than CC genotype in the Wusuli raccoon dog. There were not significant differences among litter size of the AA, BB and AB genotype caused by the G192A mutation.
3. Exon 10 and the 5'sequence of FSHR genes were cloned and sequenced in the Wusuli raccoon dog. The length of exon 10 sequence was 1398bp and it has been released to the Genbank. It's accession number was HQ 385979. The length of 5'sequence of FSHR was 757bp which included regulatory sequences and exon 1 sequence. It's Genbank accession number was HQ385977.
4. The comparison of 5'sequence and exon 10 of FSHR gene between wild and domesticated Wusuli raccoon dog showed that there were multiple mutations in the FSHR gene in the wild Wusuli raccoon dog. These mutations included 46 to 49 TTAG insertion sites,452～453AC insert,448～451 missing ACAC, and 9 SNPs in the sequence sites, while only one SNPs in the domesticated raccoon dogs. Exon 10 was highly conserved, only 3 polymorphisms was found in exon10 of FSHR in wild Wusuli raccoon dog.
5. Two polymorphic sites of FSHR gene were detected in the Wusuli raccoon with PCR-SSCP technique. They are located in the T133A of 5'regulatory region the and T623C of exon 1.
The corresponding frequencies of genotype and allele of these two polymorphisms were calculated. The correlation analysis showed that there were not significant differences between the litter size of these two SNPs in the Wusuli raccoon dog.
引文
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