动物营养学报 >

2014 , Vol. 26 >Issue 7: 1796 - 1806

DOI: https://doi.org/10.3969/j.issn.1006-267x.2014.07.011

禽营养 Poultry Nutrition

叶酸对种鸡子代胚胎期脂代谢基因表达及甲基化的影响

  • 喻小琼 ,
  • 刘冉冉 ,
  • 赵桂苹 ,
  • 郑麦青 ,
  • 李鹏 ,
  • 文杰
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  • 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193

收稿日期: 2014-02-20

  网络出版日期: 2014-07-01

基金资助

营养学国家重点实验室自主研究课题（2004DA125184G1101）；国家肉鸡产业技术体系（CARS-42）经费资助；中国农业科学院科技创新工程（ASTIP-IAS04）

收起

Effects of Maternal Folate Supplementation for Breeding Chickens on Lipid Metabolism-Related Gene Expression and Methylation in Offspring during Embryonic Development

  • YU Xiaoqiong ,
  • LIU Ranran ,
  • ZHAO Guiping ,
  • ZHENG Maiqing ,
  • LI Peng ,
  • WEN Jie
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  • State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2014-02-20

  Online published: 2014-07-01

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摘要

本试验旨在研究饲粮叶酸水平对种鸡子代胚胎期肝脏脂代谢相关基因表达及其甲基化的影响。试验选用120日龄体重相近的北京油鸡90只随机分为3组（每组30个重复，每个重复1只），单笼饲养，分别饲喂在玉米-豆粕型基础饲粮（叶酸含量0.53 mg/kg）中添加0（对照组）、2.5、5.0 mg/kg叶酸的试验饲粮，常规笼养90 d。于产蛋高峰期收集种蛋孵化，子代分别于16胚龄、19胚龄和1日龄时取肝脏组织。采用放射免疫的方法检测种蛋及饲粮叶酸含量；实时荧光定量聚合酶链式反应（RT-PCR）方法检测子代胚胎期肝脏脂肪合成关键基因mRNA的表达水平；重亚硫酸盐修饰直接测序法（BSP）和半巢式聚合酶链式反应检测肝脏固醇类调节元件结合蛋白（SREBP-1c）启动子区域（-789~-466 bp）及脂肪酸合成酶（FAS）启动子区域（-159~+143 bp）的甲基化程度。结果表明：饲粮添加2.5 mg/kg叶酸使得种蛋中叶酸沉积比对照组增加了81.92%（P0.05）；相同日龄比较，添加叶酸下调SREBP-1c基因的表达水平，且高叶酸添加组比对照组SREBP-1c甲基化程度分别下降了8.54%、9.47%及8.37%，但甲基化程度均未达到显著性差异（P>0.05）。FAS与SREBP-1c基因表达水平呈现一致的变化趋势，1日龄时达到峰值，组间达到显著差异（P<0.05），然而FAS基因在胚胎发育过程中各CpG位点均呈现高度去甲基化状态。与此不同的是，乙酰辅酶A羧化酶（ACC）与FAS基因表达水平随胚龄增加呈现相反的变化趋势，组间比较，16胚龄时差异显著（P<0.05）。由此可见，种鸡产蛋期叶酸水平在一定程度上提高种蛋中叶酸沉积（P<0.05），且影响子代胚胎部分时期脂质代谢相关基因（SREBP-1c、FAS、ACC）表达。SREBP-1c、FAS启动子区域的甲基化程度与基因表达水平没有相关性；组间比较，叶酸添加组间甲基化变化也没有差异，可能的原因是叶酸水平对该基因甲基化程度的影响不起主导作用，是影响了其他基因的甲基化程度和表达水平，间接影响SREBP-1c的基因表达水平。

关键词： 叶酸; 种鸡; DNA甲基化; 基因表达; 子代胚胎期

本文引用格式

喻小琼 , 刘冉冉 , 赵桂苹 , 郑麦青 , 李鹏 , 文杰 . 叶酸对种鸡子代胚胎期脂代谢基因表达及甲基化的影响[J]. 动物营养学报, 2014 , 26(7) : 1796 -1806 . DOI: 10.3969/j.issn.1006-267x.2014.07.011

Abstract

This experiment was conducted to investigate the effects of maternal folate supplemental level for breeding chickens on hepatic lipid metabolism-related gene expression and methylation in offspring during embryonic development. Ninety 120-day-old Beijing-you chickens with similar body weight were randomly selected and divided into 3 groups with 30 replicates in each group and each replicate contained 1 chicken, and fed in single cages. Chickens were fed corn-soybean based diet (folic acid 0.53 mg/kg) supplemented with 0 (control group), 2.5 and 5.0 mg/kg folate for 90 days, respectively. The eggs were collected and hatched when the laying rate rose to the peak. The livers were collected on days 16 and 19 of embryonic development as well as at hatching. Folate concentrations in feed and egg were determined by competitive protein binding radioassay kits. The mRNA expression levels of several hepatic lipogenesis genes were analyzed by real-time quantitative polymerase chain reaction (RT-PCR). Bisulfite sequencing-polymerase chain reaction (BSP) and semi-nested polymerase chain reaction were used to determine the CpG methylation degree at the promoter region of sterol regulator element binding protein (SREBP-1c) ranging from -789 to -466 bp and fatty acid synthase (FAS) ranging from -159 to +143 bp in liver. The results showed as follows: adding 2.5 mg/kg folate in the diet had make egg folate content 81.92% higher than that in control group (P0.05). Folate supplementation significantly decreased the transcript abundance of the SREBP-1c among groups, besides, the rates in methylation of CpG sites in SREBP-1c among three periods were decreased by 8.54%, 9.47% and 8.37% between high and low folate supplementation groups, respectively. No significant differences were found in CpG methylation of SREBP-1c promoter among all groups in different periods (P>0.05). The expression of liver FAS showed the similar tendency with SREBP-1c, and reached the peak at day 1, on which the difference among groups was significant (P<0.05). Surprisingly, the rates in methylation of CpG sites at the FAS gene promoter region were highly hypomethylated. Interestingly, the ACC mRNA expression was exactly opposite direction with FAS, and the significant effect of folate on ACC expression level was observed at the embryonic age of 16 days(P<0.05). Overall, the results indicated that folate supplementation could significantly elevate yolk folate deposition (P<0.05). Meanwhile maternal folate levels could significantly influence the transcription of some lipogenesis genes (SREBP-1c, FAS and ACC) in offspring, but the methylated regions in the promoters of SREBP-1c and FAS were not associated with gene expressions. Folate level seemed no effects on alterations in DNA methylation among groups. The results why folate status have no significant changes with CpG methylation pattern of SREBP-1c and FAS may be that folate status seems to modulate other gene expression and DNA methylation, indirectly influencing the expression of SREBP-1c.

Key words： folate; laying hen; DNA methylation; gene expression; offspring

参考文献

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