叶酸调控鸡脾和胸腺IGF2表达的表观遗传机制探究

The Study on Epigenetic Mechanism of IGF2 Expression in Spleen and Thymus Regulated by Folic Acid in Broilers

本试验旨在研究胚期注射叶酸对肉鸡脾和胸腺中IGF2基因表达的影响,并进一步探究其调控基因表达的表观遗传学机制。选择重量和大小相近的种蛋360枚,随机分为4组,在孵化期第11天(E11)分别注射0(对照)、50、100和150μg的叶酸。每个处理选取体重相近的48只1日龄雏鸡,随机分为6个重复,每个重复8只鸡,在饲养的第21和42天取胸腺和脾组织。用RT-PCR方法检测组织中IGF2的相对表达量;重亚硫酸盐修饰克隆测序法(BSP)检测IGF2启动子区域(-648/-479bp)甲基化水平;DNase I-qPCR方法检测IGF2启动子区域(-847/-616bp)染色质构象。结果表明,胚期注射100和150μg叶酸显著提高了受精蛋孵化率(P<0.05);胚期注射150μg叶酸显著提高了42日龄脾中IGF2的表达量(P<0.05)、降低了42日龄脾中该基因启动子区的甲基化水平(P<0.05),并且也显著增加了42日龄脾中IGF2启动子区的染色质可接近度(P<0.05);然而在21日龄时各叶酸处理组对脾中IGF2的表达均无显著影响(P>0.05)。与脾组织不同,150μg叶酸处理组显著提高了21日龄胸腺中IGF2的表达(P<0.05),对42日龄胸腺中该基因的表达却没有显著影响(P>0.05),并且与对照组相比,150μg叶酸处理组中21日龄胸腺组织IGF2启动子区的染色质可接近度也显著增加(P<0.05)。由此可见,在种蛋孵化第11天注射叶酸可通过影响基因启动子区的甲基化水平和染色质构象来调控组织中IGF2的表达,并且存在组织器官和时间特异性。

The study was conducted to investigate the effects of folic acid injected in incubation period on IGF2 expression in spleen and thymus in broilers,further exploring epigenetic mechanism of folic acid modulating gene expression.A total of 360 eggs,whose weight and size were mostly same,were selected and randomly assigned to 4treatments.0,50,100 and 150μg folic acid were injected into eggs,respectively on E11 of incubation.After hatching,48 healthy 1-day-old chickens selected from each treatment were divided into 6replicates and 8birds each.Spleens and thymus were collected at the age of 21 and 42d.The mRNA expression of IGF2 were tested by RT-PCR;BSP was used to determine the methylation level of IGF2 gene promoter region from-648to-479 bp and DNase I-qPCR for chromatin structure of the IGF2 promoter from-847to-616 bp.The results were shown as followed：100and 150μg folic acid groups improved hatchability of fertilized eggs（P〈0.05）;150μg folic acid injected on E11 significantly up-regulated IGF2ex-pression in spleen（P〈0.05）and reduced methylation level of gene promoter at the age of 42d（P〈0.05）;Chromatin accessibility of IGF2 promoter region was improved in 150μg folic acid group at the age of 42d（P〈0.05）in spleen.Different from spleen,150μg folic acid didn＇t affect IGF2 expression in thymus at the age of 42d（P〈0.05）but significantly increased gene expression in thymus at the age of 21d（P〈0.05）.Compared with the control group,150μg folic acid improved chromatin accessibility of IGF2 promoter in thymus at the age of 21d（P〈0.05）.Overall,the results indicated that folic acid injected on E11 could affect IGF2 expression in tissues by altering methylation level and chromatin structure of gene promoter,and there exists specificity on organs and time.