Association Between the Methylation Statuses at CpG Sites in the Promoter Region of the SLCO1B3, RNA Expression and Color Change in Blue Eggshells in Lushi Chickens
Association Between the Methylation Statuses at CpG Sites in the Promoter Region of the SLCO1B3, RNA Expression and Color Change in Blue Eggshells in Lushi Chickens
复制标题
DOI：
10.3389/fgene.2019.00161
复制
发表时间：
2019-02-26
影响因子：
3.7
通讯作者：
Kang, Xiangtao
中科院分区：
文献类型：
--
作者：
Li, Zhuanjian;Ren, Tuanhui;Kang, Xiangtao
在鸟类中发现了蓝色蛋壳特征的形成机制，SLCO1B3是调节蓝色蛋壳颜色的关键基因。将内源性逆转录病毒Eav-HP插入到SLCO1B3 5‘侧翼区，促进了SLCO1B3在鸡壳腺中的表达，这种表达导致胆盐进入壳腺，胆绿素在那里分泌到蛋壳中，形成蓝色蛋壳。然而，在同一组鸡的不同下蛋阶段，蛋壳的颜色可能会有很大的差异，蛋壳颜色变化的分子机制尚不清楚。因此，为了揭示蓝色蛋壳颜色变化的分子机理，我们分析了产蛋期蛋壳颜色的变化。结果表明，鲁氏鸡蛋壳颜色可分为三个阶段：深蓝20-25周，中蓝26-45周，浅蓝46-60周。我们进一步研究了SLCO1B3基因在8个不同周龄的表达和甲基化水平，发现SLCO1B3基因的相对表达在25和30周龄显著高于其他产蛋周龄。此外，根据硫酸氢测序聚合酶链式反应的结果，卢氏鸡SLCO1B3基因的甲基化率随着周龄的增加而逐渐增加。Pearson相关分析表明，SLCO1B3基因启动子区甲基化与蛋壳颜色和蛋壳组织中SLCO1B3的表达呈显著负相关。此外，我们预测CpG5和CpG8可能是调节SLCO1B3基因转录的关键位点。我们的发现表明，随着甲基化水平的增加，CpG5和CpG8位点的甲基化阻碍了转录因子与启动子的结合，在后期减少了SLCO1B3的表达，导致蛋壳颜色变浅。
The formation mechanism underlying the blue eggshell characteristic has been discovered in birds, and SLCO1B3 is the key gene that regulates the blue eggshell color. Insertion of an endogenous retrovirus, EAV-HP, in the SLCO1B3 5' flanking region promotes SLCO1B3 expression in the chicken shell gland, and this expression causes bile salts to enter the shell gland, where biliverdin is secreted into the eggshell, forming a blue shell. However, at different laying stages of the same group of chickens, the color of the eggshell can vary widely, and the molecular mechanism underlying the eggshell color change remains unknown. Therefore, to reveal the molecular mechanism of the blue eggshell color variations, we analyzed the change in the eggshell color during the laying period. The results indicated that the eggshell color in Lushi chickens can be divided into three stages: 20-25 weeks for dark blue, 26-45 weeks for medium blue, and 46-60 weeks for light blue. We further investigated the expression and methylation levels of the SLCO1B3 gene at eight different weeks, finding that the relative expression of SLCO1B3 was significantly higher at 25 and 30 weeks than at other laying weeks. Furthermore, the overall methylation rate of the SLCO1B3 gene in Lushi chickens increased gradually with increasing weeks of egg production, as shown by bisulfate sequencing PCR. Pearson correlation analysis showed that methylation of the promoter region of SLCO1B3 was significantly negatively correlated with both SLCO1B3 expression in the shell gland tissue and eggshell color. In addition, we predicted that CpG5 and CpG8 may be key sites for regulating SLCO1B3 gene transcription. Our findings show that as the level of methylation increases, methylation of the CpG5 and CpG8 sites hinders the binding of transcription factors to the promoter, reducing SLCO1B3 expression during the late period and resulting in a lighter eggshell color.