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

中国农业科学 ›› 2018, Vol. 51 ›› Issue (24): 4710-4719.doi: 10.3864/j.issn.0578-1752.2018.24.011

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

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

张壮彪¹(),狄冉¹,刘秋月¹,胡文萍¹,王翔宇¹,田志龙¹,张效生²,张金龙²,储明星¹()

1 中国农业科学院北京畜牧兽医研究所/农业部动物遗传育种与繁殖重点实验室,北京 100193
2 天津市畜牧兽医研究所,天津 300381

收稿日期:2018-04-18 接受日期:2018-07-07 出版日期:2018-12-16 发布日期:2018-12-16
基金资助:
国家自然科学基金(31772580);国家转基因科技重大专项(2016ZX08009-003-006和2016ZX08010-005-003);国家肉羊产业技术体系专项(CARS-38);中央级公益性科研院所基本科研业务费专项(Y2017JC24、2017ywf-zd-13);中国农业科学院科技创新工程(ASTIP-IAS13、CAAS-XTCX2016010-01-03、CAAS-XTCX2016010-03-03、CAAS-XTCX2016011-02-02);宁夏农林科学院科技创新先导资金(DWJLC-2016001);内蒙古自治区科技重大专项;农业科研杰出人才及其创新团队项目(农办人[2015]62号);国家万人计划科技创新领军人才项目(W02020274);天津市科技计划项目(16ZXZYNC00050);天津市农业科技成果转化与推广项目(201704020)

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

ZHANG ZhuangBiao¹(),DI Ran¹,LIU QiuYue¹,HU WenPing¹,WANG XiangYu¹,TIAN ZhiLong¹,ZHANG XiaoSheng²,ZHANG JinLong²,CHU MingXing¹()

1 Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture/ Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193
2 Tianjin Institute of Animal Sciences, Tianjin 300381

Received:2018-04-18 Accepted:2018-07-07 Online:2018-12-16 Published:2018-12-16

摘要/Abstract

摘要：

背景随着人们生活水平不断提高,蛋白含量丰富、胆固醇含量较少的羊肉在日常生活中越来越受青睐,羊肉总体消费需求呈逐年增长趋势。但是近年来以羊肉为主的羊产品短缺,导致羊肉的价格一直居高不下,造成羊肉供需之间的矛盾。在早期对绵羊各项生产性能研究中发现,其繁殖性能高低对羊肉生产有重要影响。因此,提高绵羊繁殖力对改变我国肉羊生产周转慢、效益差的局面有重要意义。产羔数是最重要的繁殖性状,但产羔数是一个低遗传力的数量性状,且受微效多基因的控制,故传统的育种方法难以快速改良产羔数性状。近年来随着分子标记技术的出现,研究人员发现了一些影响绵羊繁殖力的主效基因,比如BMPR1B、BMP15、GDF9等,所以后期人们开始利用常规育种结合这些分子标记进行高繁殖力绵羊品种选育。研究表明除了这些已经发现的主效基因外,仍有一些基因对绵羊的繁殖力具有一定的调控作用。目的探究影响绵羊繁殖力的候选基因BMP2、BMP6、BMP7、CAST和CART在小尾寒羊和苏尼特羊性腺轴相关组织（大脑、小脑、下丘脑、垂体、子宫、卵巢、输卵管）的表达差异,为阐明绵羊高繁殖力机理提供参考。方法以产多羔的小尾寒羊和产单羔的苏尼特羊为对象,利用实时荧光定量PCR检测上述5个基因在两个绵羊品种与性腺轴相关的7种组织中的表达差异。结果 BMP2在小尾寒羊和苏尼特羊的性腺轴7种组织中均有表达,在小尾寒羊下丘脑中的表达量高于苏尼特羊（P＜0.05）,在小尾寒羊输卵管、卵巢、垂体、小脑的表达量高于苏尼特羊（P＜0.01）,但该基因在2种绵羊的大脑和子宫中表达量差异并不显著（P＞0.05）;BMP6在小尾寒羊垂体和卵巢以及输卵管中表达量高于苏尼特羊（P＜0.01）,虽然该基因在小尾寒羊的下丘脑、子宫中的表达量高于苏尼特羊,但其表达差异并不显著（P＞0.05）;BMP7在小尾寒羊垂体、大脑的表达量高于苏尼特羊（P＜0.05）,在小尾寒羊下丘脑、输卵管、卵巢中的表达量高于苏尼特羊（P＜0.01）,但该基因在小尾寒羊和苏尼特羊的小脑和子宫中表达量差异并不显著（P＞0.05）;CAST在小尾寒羊和苏尼特羊的下丘脑、垂体中呈痕量表达,在其它组织中均有较高表达量,其在小尾寒羊输卵管和子宫中的表达量高于苏尼特羊（P＜0.01）,但在小尾寒羊和苏尼特羊大脑中的表达量几乎相同（P＞0.05）;CART在2种绵羊下丘脑中有较高表达量,在苏尼特羊垂体中的表达量高于小尾寒羊（P＜0.05）,在小尾寒羊大脑中的表达量高于苏尼特羊（P＜0.01）,而该基因在2种绵羊其它组织中的表达量差异并不显著（P＞0.05）。结论暗示这5个基因可能对绵羊繁殖力具有一定的调控作用。

关键词: 绵羊, 产羔数, 候选基因, 性腺轴, 组织表达

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

张壮彪,狄冉,刘秋月,胡文萍,王翔宇,田志龙,张效生,张金龙,储明星. 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[J]. 中国农业科学, 2018, 51(24): 4710-4719.

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

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

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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