西农萨能奶山羊ATF4基因克隆及其功能初步研究

Cloning and RNA Interference Analysis of ATF 4 Gene in Xinong Saanen Dairy Goat

转录激活子4(activating transcription factor 4,ATF4)是ATF/cAMP应答元件结合蛋白,在反刍动物的生长发育起着关键作用。本研究旨在分析西农萨能奶山羊ATF4基因序列结构,通过siRNA技术干扰ATF4基因表达,初步探究其对奶山羊乳腺上皮细胞脂质代谢和乳蛋白合成的影响。为明确ATF4在西农萨能奶山羊不同组织的表达差异,采集西农萨能奶山羊不同泌乳时期乳腺组织,克隆得到ATF4基因的CDS区并进行生物信息学分析及组织表达谱分析。结果表明,克隆得到奶山羊ATF4基因CDS区,全长为1059 bp,其编码352个氨基酸,蛋白分子量为38.54524 ku,理论等电点为4.61,有35个丝氨酸磷酸化位点,为无跨膜结构的酸性蛋白质;西农萨能奶山羊ATF4蛋白的二级结构中α-螺旋、延伸链、无规则卷曲、β-转角分别为39.77%、7.10%、51.14%和1.99%;ATF4与CEBPB、CEBPG、ATF3、FOSL2、ATF1、DDIT3、TRIB3、BTRC、CREB1、DISC 10个蛋白存在相互作用,与绵羊和牛的亲缘关系最近,同源性分别为97.45%和91.48%。组织表达谱分析结果表明,ATF4基因的表达量在瘤胃组织中最高,其次为肌肉。ATF4基因在泌乳前期表达水平最高,在泌乳盛期次之。干扰奶山羊乳腺上皮细胞ATF4基因表达,显著下调FASN、FABP3和BLG基因mRNA水平,上调PPARA、PPARG和LALBA基因mRNA水平。上述结果提示,ATF4基因可能参与奶山羊乳腺上皮细胞脂质代谢和乳蛋白合成过程。本研究为ATF4调控羊奶乳脂代谢和乳蛋白合成的分子机理提供了理论基础。

As a binding protein of ATF/cAMP response element,Transcriptional activator 4(activating transcription factor 4(ATF4)play a pivotal role in mammalian growth and development.The present study aimed to obtain the ATF4 gene sequence of Xinong Saanen dairy goats(Capra hircus),elucidate its structure and investigate its function on lipid metabolism and milk protein synthesis through siRNA interference of the ATF4 gene in goat mammary epithelial cells(GMECs).In order to clarify the expression differences of the ATF4 in different tissues of Xinong Saanen dairy goats,the breast tissues of Xinong Saanen dairy goats at different lactation stages were collected,and the CDS region of the ATF gene was cloned and analyzed by bioinformatics and tissue expression profile.The results showed that the complete CDS region of the ATF4 gene was 1059 bp,encoding 352 amino acids,the ATF4 protein is acidic protein without transmembrane structure,with a molecular weight of 38.54524 ku,a theoretical isoelectric point of 4.61,and 35 serine phosphorylation sites.The advanced structure of ATF4 protein was composed ofα-helix,extension chain irregularly coiled andβ-turn were 39.77%,7.10%,51.14%and 1.99%,respectively.ATF4 interacted with CEBPB,CEBPG,ATF3,FOSL2,ATF1,DDIT3,TRIB3,BTRC,CREB1 and DISC.Homology analysis showed that sequence homologies of Xinong Saanen dairy goat with sheep(Ovis arie s)and cattle(Bos taurus)mouse(Mus musculus)were 97.45%and 91.48%respectively.Tissue expression analysis revealed that the highest expression of TRIB3 ATF4 gene was in the rumen,followed by muscle.The expression level of the ATF4 gene was the highest in early lactation,followed by that in peak lactation.After interfering with ATF4 gene expression in mammary epithelial cells of dairy goats,the results showed that mRNA levels of FASN,FABP3 and BLG were down-regulated by interfering ATF4 significantly.Moreover,the mRNA levels of PPARA,PPARG and LALBA were significantly up-regulated by interfering ATF4.The above results suggest that ATF4 might be involved in the regulation of lipid metabolism and milk protein synthesis in GMECs.The present study provides basic data for further investigation of the molecular mechanism of ATF4 on milk fat metabolism and milk protein synthesis in dairy goats.