Cloning and Polymorphisms of 1st-7th Exons in FSHR of Jinding Ducks
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摘要: 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（185 bp）的完整序列，第1内含子（145 bp）的部分序列；序列b包含第2（75 bp）、3外显子（75 bp）、第2内含子（463 bp）的完整序列，第1（40 bp）、3内含子（47 bp）的部分序列；序列c包含第4外显子（75 bp）的完整序列，第3（180 bp）、4内含子（55 bp）的部分序列；序列d包含第5外显子（78 bp）的完整序列，第4（232 bp）、5内含子（56 bp）的部分序列；序列e包含第6（69 bp）、7外显子（75 bp）、第6内含子（117 bp）的完整序列，第5（133 bp）、7内含子（146 bp）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50 bp处存在A/G突变；在外显子2第30 bp处存在A/G突变；在外显子4第33 bp处存在C/T突变；在外显子5第45 bp处存在A/G突变，第19 bp处可能存在A/T突变，33 bp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。Abstract: 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-egg-laying Jinding ducks were compared. The fragments at the lengths of 330 bp (Fragment a), 700 bp (Fragment b), 310 bp (Fragment c), 366 bp (Fragment d) and 540 bp (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 (185 bp), as well as a partial sequence of the 1st intron (145 bp) of the gene; Fragment b had the complete sequences of the 2nd intron (463 bp), the 2nd exon (75 bp) and the 3rd exon (75 bp), in addition to the partial sequences of the 1st intron (40 bp) and the 3rd intron (47 bp); Fragment c consisted of the sequence of the 4th exon (75 bp), as well as the partial sequences of the 3rd intron (180 bp) and the 4th intron (55 bp); Fragment d included the sequence of the 5th exon (78 bp), and the partial sequences of the 4th intron (232 bp) and the 5th intron (56 bp); and, Fragment e contained the entire sequences of the 6th exon (69 bp), the 7th exon (75 bp) and the 6th intron (117 bp), also the partial sequences of the 5th intron (133 bp) and the 7th intron (146 bp). By comparison, it was found that the 1st exon had an A/G mutation at position 50 bp; the 2nd exon, an A/G mutation at position 30 bp; the 4th exon, a C/T mutation at position 33 bp; 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 FSHR polymorphism and egg-laying performance of Jinding ducks.
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Key words:
- Jinding duck /
- FSHR gene /
- clone /
- polymorphism analysis
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图 1 DNA的电泳结果
注：L1~L8为低产金定鸭个体的DNA电泳结果；H1~H9为高产金定鸭个体的电泳结果；M为DL5000。
Figure 1. Electrophoresis of DNA products
表 1 用于FSHR基因SNP筛查的引物信息
Table 1. Details of 5 primmer pairs for SNP of FSHR gene of Jinding duck
引物 引物序列 退火温度
(Tm) /℃片段大小
/bpP1 F: 5′-CAGTCATGTCACGCACAACATT-3′; R: 5′-GTGGCTGCATTTCTGCTCAC-3′ 59; 59 330 P2 F: 5′-GCATTGCTTGAAATATCTTGACT-3′; R: 5′-GCTTAGCACAGGTTAGATGGAA-3′ 56; 57 700 P3 F: 5′-GGGTATGTGGTGAAAGCCTAC-3′; R: 5′-CCCAAAATGCTATCATGAAGCT-3′ 56; 59 310 P4 F: 5′-GAAATGCTTTGCCTTTACCTT-3′; R: 5′-CAACCCATTTCTGAAGAAGGA-3′ 56; 57 366 P5 F: 5′-GTAATGCTGAGCTTCCCTGTTC-3′; R: 5′-GGCTTAATGATGAGAAAAAGGAA-3′ 58; 58 540 
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