金定鸭FSHR基因第1～7外显子的克隆及SNP位点的筛查

Cloning and Polymorphisms of 1^(st)-7^(th) Exons in FSHRof Jinding Ducks

动物的繁殖活动主要受内分泌生殖激素的调控,FSHR存在于卵泡颗粒细胞膜上,属于G蛋白偶联受体家族,对动物卵巢细胞的发育、成熟和排卵具有重要的作用。本研究以高产和低产金定鸭基因组为模板,通过PCR扩增、目的基因片段克隆和测序等方法,获取金定鸭FSHR基因1~7外显子序列,并对基因序列进行比对分析。结果显示,序列a包含第1外显子(185bp)的完整序列,第1内含子(145bp)的部分序列;序列b包含第2(75bp)、3外显子(75bp)、第2内含子(463bp)的完整序列,第1(40bp)、3内含子(47bp)的部分序列;序列c包含第4外显子(75bp)的完整序列,第3(180bp)、4内含子(55bp)的部分序列;序列d包含第5外显子(78bp)的完整序列,第4(232bp)、5内含子(56bp)的部分序列;序列e包含第6(69bp)、7外显子(75bp)、第6内含子(117bp)的完整序列,第5(133bp)、7内含子(146bp)的部分序列。根据基因序列特征,对获取的5段金定鸭FSHR基因序列进行扩增序列测序比对。结果发现:在外显子1第50bp处存在A/G突变;在外显子2第30bp处存在A/G突变;在外显子4第33bp处存在C/T突变;在外显子5第45bp处存在A/G突变,第19bp处可能存在A/T突变,33bp处可能存在C/T突变。金定鸭FSHR基因序列的克隆及SNP突变位点的发现可为后续开展FSHR基因的多态性与金定鸭产蛋性能的相关研究奠定一定的基础。

Animal reproductive activities are largely regulated by reproductive endocrine hormones.The follicle stimulating hormone receptor（FSHR）is found in the follicular granulose membrane of an ovary cell in a female animal.It is a G protein coupled receptor that plays an important role in the development,maturation and ovulation of the ovary cells.Considering its critical association with poultry broodiness,FSHRs from high-and low-egglaying Jinding ducks were compared.The fragments at the lengths of 330bp（Fragment a）,700bp（Fragment b）,310bp（Fragment c）,366bp（Fragment d）and 540bp（Fragment e）of the genes from the genomic DNAs extracted from the blood samples of the ducks were amplified by PCR.The sequence analysis showed that Fragment a included the entire sequence of the 1st exon（185bp）,as well as a partial sequence of the 1st intron（145bp）of the gene;Fragment b had the complete sequences of the 2nd intron（463bp）,the 2nd exon（75bp）and the 3rd exon（75bp）,in addition to the partial sequences of the 1st intron（40bp）and the 3rdintron（47bp）;Fragment c consisted of the sequence of the 4th exon（75bp）,as well as the partial sequences of the 3rdintron（180bp）and the 4thintron（55bp）;Fragment d included the sequence of the 5th exon（78bp）,and the partial sequences of the 4thintron（232bp）and the 5th intron（56bp）;and,Fragment e contained the entire sequences of the 6th exon（69bp）,the 7th exon（75bp）and the 6thintron（117bp）,also the partial sequences of the 5thintron（133bp）and the 7thintron（146bp）.By comparison,it was found that the 1st exon had an A/G mutation at position 50bp;the 2nd exon,an A/G mutation at position 30bp;the 4th exon,a C/T mutation at position 33bp;and,the 5th exon,an A/G mutation at position 45 bp,an A/T mutation at 19 bp,as well as a C/T mutation at 33 bp.The results obtained would pave the way for further study on the FSHRpolymorphism and egg-laying performance of Jinding ducks.