5个基因在小尾寒羊和苏尼特羊性腺轴相关组织中表达分析

doi:10.3864/j.issn.0578-1752.2018.24.011

Abstract

Abstract:

【Background】With the continuous improvement of living standard, mutton with rich protein and low cholesterol content is increasingly favored in daily life, and the overall consumption demand of mutton is increasing year by year. However, in recent years, the shortage of mutton-based sheep products has caused the price of mutton to remain high, resulting in a contradiction between supply and demand of mutton. In the early researches on the performance of sheep, it was found that the reproductive performance had an important impact on the mutton production. Therefore, improving the fecundity of sheep is of great significance for changing the situation of slow turnaround and poor efficiency of meat sheep production in China. The litter size is the most important reproductive trait, but the litter size is a quantitative trait of low heritability and is controlled by micro-multiple genes. Therefore, traditional breeding methods are difficult to rapidly improve litter size. In recent years, with the advent of molecular marker technology, researchers have discovered some major genes that affect the fertility of sheep, such as BMPR1B, BMP15, GDF9 and other genes, afterwards, researches began to use conventional breeding methods combined with these molecular markers to cultivate new sheep breeds with high fecundity. Studies have shown that in addition to these major genes that have been discovered, there are still some genes that have a certain regulatory effect on the fecundity of sheep. Based on this situation, the purpose of this study was to explore the differential expression of candidate genes which may affect the fertility of sheep. 【Objective】 BMP2, BMP6, BMP7, CAST and CART, in the tissues associated with the gonadal axis (brain, cerebellum, hypothalamus, pituitary, uterus, ovary and oviduct) in Small Tail Han sheep and Sunite sheep, which would provide a reference for clarifying the mechanism of high fecundity of sheep. 【Method】 Polytocous Small Tail Han sheep and monotocous Sunite sheep were used as the experimental animals, and real-time fluorescence quantitative PCR was performed to detect the expression difference of these five genes in seven gonadal-related tissues in two sheep breeds. 【Result】 The results showed that BMP2 gene was expressed in all seven tissues of the gonadal axis. The expression of BMP2 in hypothalamus in Small Tail Han sheep was higher than that in Sunite sheep (P＜0.05). The expression of BMP2 in oviduct, ovary, pituitary and cerebellum in Small Tail Han sheep was higher than that in Sunite sheep (P＜0.01), however, the expression difference of this gene in the brain and uterus of the two sheep breeds was not significant (P＞0.05). The expression of BMP6 in pituitary, ovary and oviduct in Small Tail Han sheep was higher than that in Sunite sheep (P＜0.01), although the expression level of this gene in the hypothalamus and uterus of Small Tail Han sheep was higher than that of Sunite sheep, however, the expression difference was not significant (P＞0.05). The expression of BMP7 in pituitary and brain of Small Tail Han sheep was higher than that in Sunite sheep (P＜0.05), the expression of BMP7 in hypothalamus, oviduct and ovary of Small Tail Han sheep was higher than that of Sunite sheep (P＜0.01), but there was no significant difference in the expression of this gene in the cerebellum and uterus of Small Tail Han sheep and Sunite sheep (P＞0.05). The trace expression of CAST was found in hypothalamus and pituitary, and the higher expression in other tissues in two sheep breeds, the expression of CAST gene in oviduct and uterus of Small Tail Han sheep was higher than that of Sunite sheep (P＜0.01), however, the expression levels of this gene in the brains of Small Tail Han sheep and Sunite sheep were almost the same (P＞0.05). The CART was highly expressed in hypothalamus of two sheep breeds. The expression of CART gene in pituitary of Sunite sheep was higher than that of Small Tail Han sheep (P＜0.05), and the expression of CART in brain of Small Tail Han sheep was higher than that of Sunite sheep (P＜0.01), whereas, there was no significant difference in the expression of this gene in other tissues used in this experiment of two sheep breeds (P＞0.05). 【Conclusion】 These results implied that the five genes may have some regulatory roles on sheep fertility.

Key words: sheep, litter size, candidate genes, gonadal axis, tissue expression

ZHANG ZhuangBiao,DI Ran,LIU QiuYue,HU WenPing,WANG XiangYu,TIAN ZhiLong,ZHANG XiaoSheng,ZHANG JinLong,CHU MingXing. Expression Analysis of Five Genes in the Gonadal Axis of Small Tail Han Sheep and Sunite Sheep[J].Scientia Agricultura Sinica, 2018, 51(24): 4710-4719.

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试剂 Reagent	各试剂加入量 Volume of each reagent
PrimeScript 反转录混合引物Ⅰ PrimeScript RT Enzyme Mix Ⅰ	1.0 μL
Oligo dT引物 Oligo dT Primer	1.0 μL
随机引物 Random 6 mers	1.0 μL
5×PrimeScript荧光定量缓冲液 5×PrimeScript Buffer (for Real Time)	4.0 μL
RNA	1.0 μg
双蒸水 RNase-Free ddH₂O	12μL

试剂 Reagent	各试剂加入量 Volume of each reagent (μL)
荧光定量Ex Taq引物Ⅱ SYBR Premix Ex TaqⅡ	10.0
上游引物（10 μmol·L^-1） Upstream primer (10 μmol·L^-1)	0.8
下游引物（10 μmol·L^-1） Downstream primer (10 μmol·L^-1)	0.8
cDNA 模板（500 ng·μL^-1） cDNA template (500 ng·μL^-1)	2.0
双蒸水 RNase-Free ddH₂O	6.4

Expression Analysis of Five Genes in the Gonadal Axis of Small Tail Han Sheep and Sunite Sheep

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基因名称 Name	引物序列 Primer sequence	片段大小 Length (bp)	退火温度 Tm (℃)
BMP2	F:5′-ATCACCTGAACTCCACGAA-3′	140	60
BMP2	R: 5′-TACCACCTTCTCATTCTCATC-3′	140	60
BMP6	F:5′- AGCAGACTACAACAGCAGCGA-3′	267	60
BMP6	R:5′- AGCACCGAGATGGCGTTCA-3′	267	60
BMP7	F:5′- AAAACAGCAGCAGCGACCAGAG-3′	123	68
BMP7	R:5′- CCTCACAGTAGTAGGCGGCATAGC-3′	123	68
CAST	F:5′-TGGGGCCCAATGACGCCATCGATG - 3′	222	60
CAST	R:5′- GGTGGAGCAGCACTTCTGATCACC - 3′	222	60
CART	F:5′- AGCAGCCACCATCAGAGAAA - 3′	214	60
CART	R:5′- TCCAGAGCAGTATCCATGTCT - 3′	214	60
β-actin	F:5′- CCAACCGTGAGAAGATGACC-3′	97	60
β-actin	R:5′- CCCGAGGCGTACAGGGACAG-3′	97	60

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