关中奶山羊CSN1S1基因克隆、生物信息学及组织表达谱分析

doi:10.16431/j.cnki.1671-7236.2022.03.007

摘要/Abstract

摘要： 【目的】通过分析关中奶山羊αs1-酪蛋白（alpha-s1 casein，CSN1S1）基因组织表达谱及其生物信息学功能，初步探究CSN1S1基因在奶山羊乳成分合成中发挥的作用。【方法】以关中奶山羊为研究对象，利用PCR扩增并克隆CSNIS1-1和CSN1S1-2 2个突变形态，并利用ProtParam、NetPhos、SingalP 4.1 Server、NPS和Phyre2等多种生物信息软件和在线工具对CSN1S1基因及其突变形态的蛋白质结构、理化性质、磷酸化位点等进行分析，通过实时荧光定量PCR检测CSNIS1-1和CSN1S1-2在关中奶山羊肝脏、脾脏、乳腺、肾脏、子宫、输卵管6个组织中的相对表达量。【结果】关中奶山羊乳腺上皮细胞中存在CSN1S1-1和CSN1S1-2 2种突变形态。对测序结果比对显示，CSN1S1-1存在3个碱基的突变，CSN1S1-2不仅存在3个碱基的突变，还存在6个碱基的缺失。CSN1S1-1与CSN1S1蛋白相似性为99.07%，CSN1S1-2与CSN1S1蛋白相似性为97.20%。蛋白理化性质分析显示，CSN1S1-1中碱基的突变导致第31位亮氨酸变成脯氨酸、第92位谷氨酰胺变成谷氨酸；CSN1S1-2除上述改变之外，第83位由丝氨酸变成半胱氨酸、第84位由谷氨酸变成谷氨酰胺，还存在第81和82位2个丝氨酸的缺失。实时荧光定量PCR结果显示，CSN1S1-1和CSN1S1-2在关中奶山羊各组织中相对表达趋势基本相同，其中在乳腺、子宫、输卵管中相对表达量较高，在肝脏、脾脏和肾脏中基本不表达，在子宫中CSN1S1-2的表达量显著高于CSN1S1-1（P<0.05）。【结论】关中奶山羊乳腺上皮细胞中存在CSN1S1-1和CSN1S1-2 2种突变形态，且2种突变形态与CSN1S1的蛋白相似性均达到97%以上。CSN1S1-1在蛋白结构上与CSN1S1已有明显区别，而CSN1S1-2中6个碱基的缺失是导致氨基酸改变、磷酸化位点缺失与移位的直接原因。CSN1S1-2在子宫中表达显著高于CSN1S1-1，其可能在子宫中发挥潜在功能，还有待进一步探索。

关键词: 关中奶山羊; CSN1S1基因; 克隆; 组织表达谱; 生物信息学分析

Abstract: 【Objective】 By constructing the tissue expression profile and bioinformatics function of alpha-s1 casein(CSN1S1)gene in Guanzhong dairy goats, the function of CSN1S1 gene in milk composition synthesis of Guanzhong dairy goats was preliminarily investigated.【Method】 Guanzhong dairy goats were taken as the research object, two mutant forms of CSN1S1 were cloned which were named CSNIS1-1 and CSN1S1-2. The protein structure, physicochemical property and phosphorylation sites of CSN1S1 gene and its mutant form were analyzed using a variety of biological information softwares and online tools such as ProtParam, NetPhos, SingalP 4.1 Server, NPS and Phyre2. The relative expression of CSNIS1-1 and CSN1S1-2 in liver, spleen, mammary gland, kidney, uterus and fallopian tube of Guanzhong dairy goats were detected by Real-time quantitative PCR.【Result】 The results showed that two mutant forms CSN1S1-1 and CSN1S1-2 were found in mammary epithelial cells of Guanzhong dairy goats. Sequencing results showed that compared with CSN1S1 sequence, 3 bases mutations were found in CSN1S1-1 and CSN1S1-2, in addition, there were total 6 bases deletions in two positions of CSN1S1-2. The similarity was 99.07% between CSN1S1-1 and CSN1S1, and 97.20% between CSN1S1-2 and CSN1S1.The analysis of protein physicochemical properties showed that the mutation of bases in CSN1S1-1 resulted in the change of leucine at position 31 to proline and glutamine at position 92 to glutamic acid. In addition to the above changes, CSN1S1-2 changed from serine to cysteine at position 83, and changed from glutamic acid to glutamine at position 84. In addition, there were deletions of two serines at positions 81 and 82. Real-time quantitative PCR results showed that the relative expression trends of CSN1S1-1 and CSN1S1-2 in Guanzhong dairy goat tissues were basically the same, and the relative expression levels were higher in mammary gland, uterus and fallopian tube, and basically not expressed in liver, spleen and kidney. The expression of CSN1S1-2 in uterus was significantly higher than that of CSN1S1-1 (P<0.05).【Conclusion】 Two mutant forms CSN1S1-1 and CSN1S1-2 were found in the mammary epithelial cells of Guanzhong dairy goats, and the protein similarity between the two mutant forms and CSN1S1 reached more than 97%. CSN1S1-1 was obviously different from CSN1S1 in protein structure, and the deletion of 6 bases in CSN1S1-2 might be the direct cause of amino acid change, deletion and displacement of phosphorylation site. The expression of CSN1S1-2 in uterus was significantly higher than that of CSN1S1-1, and its potential function in uterus remains to be further explored.

Key words: Guanzhong dairy goats; CSN1S1 gene; cloning; tissue expression profile; bioinformatics analysis

中图分类号:

侯金星, 王战航, 朱俊儒, 江悦, 刘淑娟, 安小鹏. 关中奶山羊CSN1S1基因克隆、生物信息学及组织表达谱分析[J]. 中国畜牧兽医, 2022, 49(3): 857-865.

HOU Jinxing, WANG Zhanhang, ZHU Junru, JIANG Yue, LIU Shujuan, AN Xiaopeng. Cloning, Bioinformatics and Tissue Expression Profile Analysis of CSN1S1 Gene in Guanzhong Dairy Goats[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 857-865.

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