阉割对金华猪肝脏miR-122和miR-378表达量和膻味性状的影响

摘要

microRNA是一种小分子RNA,是细胞内复杂而精确的调控网络的组成部分.为了研究阉割对miR-122和miR-378表达量的影响以及miR-122和miR-378对雄烯酮和粪臭素代谢的调控作用,本研究利用荧光定量PCR检测了miR-122和miR-378在不同生长阶段金华猪(Sus scrofa)公猪肝脏中的表达量变化及其在阉割和非阉割公猪体内表达量的差异,利用高效液相色谱法(high performance liquid chromatography,HPLC)检测了金华猪皮下脂肪的粪臭素含量,并预测了调控pre-miR-122和pre-miR-378转录的相关转录因子及miR-122和miR-378与雄烯酮、粪臭素代谢相关基因的靶关系.结果发现,miR-122在胚胎期高表达,随着日龄的增加表达量逐渐下降;miR-378在胚胎期高表达,生长期呈现先增后减的态势.阉割后两者的表达量均较同期非阉割组表达下调.并且阉割后皮下脂肪中粪臭素的含量显著下降(P＜0.01).根据研究结果推测,阉割后激素水平的变化通过相关转录因子影响microRNA的表达,直接或间接影响雄烯酮和粪臭素代谢而实现对公猪膻味性状的调控.而在这个调控网络中,microRNA可能发挥了重要作用,为深入研究公猪膻味性状提供了一个新的思路.%MicroRNA(miRNA) is a class of small RNA,it is involved in the intracellular complicated and precise regulatory networks.In order to study the effect of castration on the expression of miR-122 and miR-378 and the regulation effect of miR-122 and miR-378 on androstenone and skatole metabolism,we detected the skatole content in subcutaneous fat of boars and barrows with the help of high performance liquid chromatography (HPLC) and the expression of miR-122 and miR-378 in various growth stages in liver of Jinhua Pig (Sus scrofa) by quantitative Real-time PCR (qRT-PCR) and analysed the expression variation between boars and barrows.The results showed that the skatole content in adipose tissue was higher (P＜0.01) in boars (mean 87.21 μg/kg,n=9) than that in barrows (79.87 μg/kg,n=9),and there was a significance change of the expression of miR-122 and miR-378 between embryonic and growth period.In the embryonic period the expression of miR-122 increased firstly and lasted to the 80th day,and then decreased.In the growth period the expression of miR-122 was decreased gradually.With the development of embryonic liver the expression of miR-378 continued to increase,while in the growth period miR-378 increased firstly and then decreased,and at 60 d reached the maximum.After 60 d the expression of both miR-122 and miR-378 of boars and barrows decreased significantly (P＜0.01) with the increase of day age.At 60 and 120 d the expression of miR-122 of barrows were significantly decreased (P＜0.01) by comparison to boars,and at 90th day it was also decreased significantly (P＜0.05).After castration,the expression of miR-378 was decreased significantly (P＜0.01) at 60,90 and 120 d.We also predicted the transcription factors which may affect the transcription of pre-miR-122 and pre-miR-378,and the target genes which may affect the androstenone and skatole metabolism.We found that miR-122 and miR-378 had target relationship with androstenone and skatole metabolism genes SULT1A1 (sulfotransferase family,cytosolic,1A,phenol-preferring,member 1),SULT2A1(sulfotransferase family,cytosolic,2A,dehydroepiandrosterone (DHEA)-preferring,member 1) and CYP2E1(cytochrome P450,family 2,subfamily E,polupeptide 1).We forecasted that the upstream sequence of 5.2～4.9 kb of pre-miR-122 and the upstream sequence of 2.2～1.8 kb of pre-miR-378 were the promoter region,and there were related transcription factor binding sites.In the upstream sequence of pre-miR-378,there was a CpG island close to the promoter region,which indicated that the transcription of pre-miR-378 might be affected by methylation.In conclusion,we suggested that the changes of hormone level after castration may affect the expression of microR-NA through the relevant transcription factors,and then affect the boar taint by regulating the androstenone and skatole metabolism directly or indirectly.And microRNAs may play an important role in the regulatory networks.All in all,we provide a new idea for further research on boar taint.