贵州黑山羊ADD1基因克隆、生物信息学及组织表达规律研究

Cloning,bioinformatics and tissue expression patterns of ADD1 gene in Guizhou black goats

为了分析贵州黑山羊ADD1基因编码区序列(coding sequence, CDS)信息及在组织中的表达规律,试验克隆了贵州黑山羊ADD1基因CDS并进行了生物信息学分析,同时分析了该基因在12月龄贵州黑山羊心脏、肝脏、脾脏、肺脏、肾脏、皮下脂肪、背最长肌和腿肌组织中的表达规律,以及在1,3,6,9,12月龄贵州黑山羊皮下脂肪、背最长肌和腿肌组织中的表达时序情况。结果表明:贵州黑山羊ADD1基因CDS大小为2 304 bp,编码767个氨基酸,与山羊ADD1基因序列相似性较高,与禽类的相似性较低;ADD1蛋白中的主要氨基酸为丝氨酸(Ser)、脯氨酸(Pro)、谷氨酸(Glu)和亮氨酸(Leu),含量分别为9.0%、8.7%、8.5%、8.2%,分子质量为84.05 ku,分子式为C_(3682)H_(5840)N_(1036)S_(24),理论等电点为5.74,为不稳定蛋白和亲水蛋白,主要定位在细胞核中,与ADD蛋白家族(ADD2和ADD3)、加帽蛋白(capping actin protein, CAPG)、肌侵蛋白(myotrophin, MTPN)、X盒结合蛋白1(X-box binding protein 1,XBP1)、固醇调节元件结合蛋白1(sterol-regulatory element-binding protein 1,SREBP1)等蛋白存在相互作用,二级结构和三级结构中主要含有α螺旋和无规则卷曲;ADD1基因在12月龄贵州黑山羊心脏、肝脏、脾脏、肺脏、肾脏、皮下脂肪、背最长肌和腿肌组织中均有表达,皮下脂肪中的表达量最高,极显著高于其他组织(P<0.01),其次在心脏、背最长肌、肝脏和腿肌中表达量较高,在脾脏中表达量最低。ADD1基因在1,3月龄贵州黑山羊脂肪、背最长肌和腿肌组织中的表达量均较低。在脂肪组织中6月龄时最高,极显著高于其他月龄(P<0.01);3月龄时最低,极显著低于其他月龄(P<0.01)。在背最长肌中9,12月龄较高,二者差异不显著(P>0.05),但均极显著高于其他月龄(P<0.01);6月龄表达量最低,与1月龄差异不显著(P>0.05),但二者均极显著低于其他月龄(P<0.01)。在腿肌中9月龄时最高,极显著高于其他月龄(P<0.01);12月龄最低,与1月龄差异不显著(P>0.05),但二者均极显著低于其他月龄(P<0.01)。说明ADD1基因在贵州黑山羊各组织中的表达具有广谱性和差异性。

In order to analyze the coding sequence(CDS)information and expression patterms of the ADDl gene in Guizhou black goats,the CDS region of the ADD1 gene in Guizhou black goats was cloned and subjected to bioinformatics analysis.The tissue expression patterns of ADD1 gene in hearts,livers,spleens,lungs,kidneys,subcutaneous fat,longissimus dorsi,and leg muscles of Guizhou black goats at 12 months,as well as the temporal expression patterns in the subcutaneous fat,longissimus dorsi,and leg muscle tissues of Guizhou black goats at 1,3,6,9,and 12 months old were analyzed.The results showed that the CDS region of the ADD1 gene in Guizhou black goats was 2304 bp long,encoding 767 amino acids.The ADD1 gene sequence had high similarity to goats and low similarity to birds.The main amino acids in the ADD1 protein were serine(Ser),proline(Pro),glutamic acid(Glu),and leucine(Leu),accounting for 9.0%,8.7%,8.5%,and 8.2%respectively.The molecular weight was 84.05 ku,with a molecular formula of C3682Hs840 N1036S24,and a theoretical isoelectric point of 5.74.It was an unstable and hydrophilic protein,mainly located in the cell nucleus.It interacted with other proteins such as the ADD protein family(ADD2 and ADD3),capping actin protein(CAPC),myotrophin(MTPN),X-box binding protein 1(XBP1),and sterol-regulatory element-binding protein 1(SREBP1).The secondary and tertiary structures mainly consisted ofα-helices and irregular coils.The ADD1 gene was expressed in the hearts,livers,spleens,lungs,kidneys,subcutaneous fat,longissimus dorsi,and leg muscle tissues of Guizhou black goats at 12 months.The expression level was the highest in subcutaneous fat,which was significantly higher than that in other tissues(P<O.01),followed by the higher expression level in the hearts,longissimus dorsi,livers,and leg muscle tissues,and the lowest expression level in the spleen.The expression level of the ADD1 gene in the fat,longissimus dorsi,and leg muscle tissues of Guizhou black goats at 1 and 3 months old was low.In the fat tissue,the expression level was the highest at 6 months old,which was significantly higher than that at other ages(P<0.01),and the lowest level was found at 3 months old,which was significantly lower than that at other ages(P<0.01).In the longest back muscle,the expression level was higher at 9 and 12 months,with no significant difference between them(P>0.05),but both were significantly higher than that at other ages(P<0.01).The expression level was the lowest at 6 months old,and there was no isignificant difference from 1 month old(P>0.05),but both were significantly lower than that at other ages(P<0.01).In the leg muscle,the expression level was the highest at 9 months old,which was significantly higher than that at other ages(P<0.01),and the lowest at 12 months old,with no significant difference from 1 month old(P>0.05),but both were significantly lower than that at other ages(P<0.01).These findings indicated that the ADD1 gene was expressed in a wide range of tissues in Guizhou black goats,and the expression levels was different among different tissues.