中国农业科学 ›› 2017, Vol. 50 ›› Issue (14): 2816-2825.doi: 10.3864/j.issn.0578-1752.2017.14.017

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

山羊早期胎儿组织TET1与Wnt通路基因的表达变化及其相关性

谭强,罗南剑,张艳丽,安炳星,陈秋羊,赵永聚   

  1. 西南大学动物科技学院/重庆市牧草与草食家畜重点实验室/重庆市草食动物资源保护与利用工程技术研究中心,重庆 400715
  • 收稿日期:2016-09-01 出版日期:2017-07-16 发布日期:2017-07-16
  • 通讯作者: 赵永聚,Tel:023-68251255;E-mail:zyongju@163.com
  • 作者简介:谭强,Tel:18883394145;E-mail:tq19952010@126.com。罗南剑,Tel:13996488708;E-mail:luonanjian28@sina.cn。谭强和罗南剑为同等贡献作者。
  • 基金资助:
    国家级大学生创新创业训练计划(201510635013)、重庆高校创新团队建设计划资助项目(CXTDG201602004)、中央高效基本业务费专项资金(XDJK2017A003)、重庆市社会民生科技创新专项项目(cstc2016shmszx80064)

Expression Patterns and Correlation of Wnts and TET1 Genes in Early Fetal Tissues of Dazu Black Goat

TAN Qiang, LUO NanJian, ZHANG YanLi, AN BingXing,CHEN QiuYang, ZHAO YongJu   

  1. College of Animal Science and Technology, Southwest University/Chongqing Key Laboratory of Forage & Herbivore / Chongqing Engineering Research Center for Herbivores Resource Protection and Utilization, Chongqing 400715
  • Received:2016-09-01 Online:2017-07-16 Published:2017-07-16

摘要: 【目的】通过检测TET1和Wnt信号通路相关基因以及DKK家族基因在山羊胎儿发育早期的表达变化,分析TET1与Wnt通路基因的相关性,为TET1调控山羊胎儿发育研究提供理论依据。【方法】选取12只健康大足黑山羊母羊,自然发情后与同一只种公羊自然交配。采用剖腹产手术的方法,分别获得妊娠20、25、30、60和90d的胎儿,对胎儿的生长指标(体重、体长)进行统计,并采集了60和90d胎儿的组织器官样品(心、肝、肺、肾、脑、皮肤),通过Real-time PCR(RT-PCR)检测各样品中TET1基因,DKK家族基因(DKK1、DKK2、DKK3)和Wnt家族基因(Wnt2、Wnt2b、Wnt4、Wnt5a、Wnt5b、Wnt7b、Wnt16)的相对表达量。利用SPSS软件分析山羊胎儿发育早期不同阶段TET1与WNT信号通路相关基因相关性以及基因表达显著性(P<0.05)。【结果】山羊妊娠早期胎儿生长在60d后有显著变化。荧光定量检测结果表明,TET1基因表达随妊娠天数的增加呈上升趋势。Wnt家族基因在山羊胎儿发育中都检测到表达(Wnt2,-2b,-4,-5a,-5b,-7b,-16)。其中,Wnt2和Wnt7b表达量随胎儿发育逐渐增高;Wnt2b、Wnt5a、Wnt5b、Wnt7b在妊娠30 d时有显著高表达(P<0.05);Wnt4在胎儿发育20 d时表达显著(P<0.05);Wnt16基因在妊娠25 d有显著高表达(P<0.05)。DKK家族基因表达检测结果显示,DKK1在胎儿发育早期阶段都有表达,DKK2/3在妊娠初期表达量较低,后期表达增高。通过组织中基因表达检测显示,TET1在90d胎儿肝、肺、肾和脑中的表达水平相比于60d胎儿组织升高,肝中表达量显著(P<0.05)。Wnt家族基因Wnt2在组织器官中有相对活跃的表达,妊娠90d胎儿肺中表达量极显著(P<0.01);Wnt16基因在胎儿皮肤组织中表达显著(P<0.05),且维持在一个较高的水平;Wnt5a和Wnt7b在肾中表达显著(P<0.05),其他Wnt基因在组织中都有表达。相关性分析显示,胎儿生长指标(体重、体长)变化与TET1的表达呈极显著正相关(P<0.01);TET1在胎儿发育早期的表达与Wnt2、Wnt7b、Wnt16呈现正相关,与Wnt2b、Wnt4、Wnt5a、Wnt5b呈负相关,其中与Wnt5b呈显著负相关(P<0.05),与Wnt7b呈极显著正相关(P<0.01)。Wnt通路基因之间也有相互关系,Wnt2与Wnt4呈极显著负相关(P<0.01)。Wnt2与Wnt7b,Wnt2b与Wnt5a、Wnt5b,Wnt5a与Wnt5b呈极显著正相关(P<0.01)。Wnt4与Wnt5a呈显著正相关(P<0.05)。【结论】获得了TET1与Wnt基因在山羊胎儿发育早期的表达模式,并进行了相关性分析,填补了这些基因在山羊方面的研究空白;TET1与Wnt基因对山羊胎儿早期的发育和组织的形成是一个动态的调控变化过程;TET1基因表达与部分Wnt基因呈现显著正相关,部分呈现显著负相关;Wnt通路基因之间表达量呈现一定的相关性。这些数据为TET1与Wnt分子调控山羊早期胎儿发育的机制深入研究提供了参考。

关键词: 山羊胎儿, TET1, Wnt通路基因, 表达变化, 相关性

Abstract: 【Objective】The aim of the research was to quantitate the relative expression level of TET1, Wnt and DKK genes during early fetus development in goat, analyze the correlation between TET1 and Wnts genes in further. 【Method】Twelve healthy Dazu black goats copulated with the same ram by wild pairing while these goats with spontaneous estrus. Caesarean section was used to obtain fetus that pregnancy on days 20, 25, 30, 60 and 90, respectively, the growth indexes of the fetus (fetal weight, fetal length) were tested, and the tissue samples of 60d and 90d (fetal heart, liver, lung, kidney, brain and skin) were collected. Real-time (RT-PCR) was used to detect the relative expression level of TET1 gene, DKK family genes (DKK1, DKK2, DKK3) and Wnt family genes (Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt5b, Wnt7b, Wnt16). SPSS software was used to analyze the correlation between TET1 and Wnt signaling pathway genes (P<0.05) at different stages of fetal development. 【Result】 According to the statistics, there were significant changes in fetal development in early pregnancy after 60d. The results showed that the expression of TET1 gene increased with the development of gestational days. All Wnt family genes were detected in the fetus (Wnt2, -2b, -4, -5a, -5b, -7b, -16). Among these changes of fetal development, Wnt2 and Wnt7b expression increase with fetal development gradually; Wnt2b, Wnt5a, Wnt5b, and Wnt7b genes has a significant highly expression on pregnancy 30d (P<0.05); the expression of Wnt4 was significantly in fetus on pregnancy 20d (P<0.05); Wnt16 gene was significantly in fetus on pregnancy 25d (P<0.05). The expression level of DKK family genes showed that DKK1 was detected in the early pregnancy of fetal development, and DKK2/3 performed a low expression level in the early pregnancy, whereas increased in later stage. The expression level of TET1 in liver, lung, kidney and brain in 90d fetus was higher than 60d fetal tissue, there was a significantly expression in liver (P<0.05). Interestingly, Wnt2 had a highly significant expression level in all tissues of fetus on pregnancy 60 and 90d, especially in lung (P<0.01); Wnt16 had a highly expression in fetal skin (P<0.05); Wnt5a and Wnt7b had a significantly expression level in kidney (P<0.05), other Wnt genes of Wnt family were detected in fetal tissues as well. Correlation analysis showed that the fetal growth indexes (fetal weight, body length) were positively correlated to changes in the expression of TET1 (P<0.01); TET1 in early fetal development and the expression of Wnt2, Wnt7b and Wnt16 gene showed a positive correlation, and negatively correlated with Wnt2b, Wnt4, Wnt5a and Wnt5b, which was negatively correlated with Wnt5b (P<0.05), and significantly and positively correlated with Wnt7b (P<0.01). There was also a correlation between Wnt pathway genes, Wnt2 and Wnt4 showed a significant negative correlation (P<0.01). Wnt2 and Wnt7b, Wnt2b and Wnt5a, Wnt5b, Wnt5a and Wnt5b showed a significant positive correlation (P<0.01). Wnt4 was positively correlated with Wnt5a (P<0.05). 【Conclusion】 The dynamic expression patterns of TET1 and Wnt genes in early fetal development of goat, and correlation of these genes were obtained from this study which fill the blank of those genes in this research field. The article revealed TET1 and Wnts regulate the embryonic development and the formation of tissues by a dynamic pattern. TET1appeared a positively correlation with some Wnt genes while negatively with others. It is also showed interactive in Wnt signaling pathway genes. The data obtained from the research will provide a reference for molecular mechanism research of TET1 and Wnts.

Key words: goat fetus, TET1, Wnt signaling pathway, expression pattern, correlation