水牛BTG2基因序列分析、过表达载体构建及组织表达研究

doi:10.16431/j.cnki.1671-7236.2025.09.001

摘要/Abstract

摘要： 【目的】克隆水牛B细胞易位基因2(B-cell translocation gene 2，BTG2)基因编码区，分析其编码蛋白的生物学特性，构建过表达载体并转染水牛卵巢颗粒细胞，探究该基因在水牛不同组织中的表达模式，为后续深入研究水牛BTG2基因的生物学功能奠定基础。【方法】采集水牛心脏、肝脏、脾脏、肺脏、肾脏、小肠、瘤胃、子宫角、乳腺和卵巢组织，提取总RNA后反转录获得cDNA。以水牛卵巢cDNA为模板克隆水牛BTG2基因编码区并测序，对水牛及不同物种的BTG2基因进行序列比对和系统进化树构建，利用在线软件预测水牛BTG2蛋白的理化性质、信号肽、跨膜结构等。构建OE-BTG2过表达载体并转染水牛颗粒细胞，检测转染后BTG2基因的表达情况。通过实时荧光定量PCR检测BTG2基因在水牛不同组织中的表达情况。【结果】水牛BTG2基因CDS区序列全长453 bp，共编码150个氨基酸。相似性比对结果显示，水牛与牦牛、普通牛、山羊和绵羊的核苷酸序列相似性均＞98%，其中与牦牛和普通牛的相似性最高，达99.8%。系统进化树显示，水牛与牦牛和普通牛的遗传距离最近，与家鼠的遗传距离最远。水牛BTG2蛋白为亲水性蛋白，无跨膜结构和信号肽，氨基酸序列的第1―108位有1个btg1结构域，共有15个磷酸化位点和1个O-糖基化位点，主要定位于细胞核中。BTG2蛋白主要与CNOT7、CNOT8、CNOT11、CNOT9、CNOT1、CNOT10、ATF3、ANKRD49、HES6和PRMT1等蛋白互作。水牛BTG2蛋白二级结构以α-螺旋、延伸链和无规则卷曲为主，三级结构预测结果与其一致。成功构建OE-BTG2过表达载体并转染至水牛颗粒细胞，与对照组(pcDNA3.1(+))相比，过表达组(OE-BTG2)细胞中BTG2基因表达量极显著升高(P＜0.01)。实时荧光定量PCR结果显示，BTG2基因在水牛的心脏、肝脏、脾脏、肺脏、肾脏、小肠、瘤胃、子宫角、乳腺和卵巢组织中均有表达，其中在肝脏中的表达量显著高于其他组织(P＜0.05)。【结论】 BTG2基因在不同物种中具有较高的保守性，且在水牛不同组织中广泛表达，其中在肝脏中表达量最高。试验结果为深入研究水牛BTG2基因的功能提供了参考依据。

关键词: 水牛; BTG2基因; 生物信息学; 过表达载体; 组织表达

Abstract: 【Objective】 The aim of this study was to clone the CDS sequence of B-cell translocation gene 2(BTG2)gene in buffalo,analyze the biological characteristics of its encoded protein,construct the overexpression vector and transfect buffalo granulosa cells,and explore the expression patterns of BTG2 gene in different tissues of buffalo,laying theoretical basis for the subsequent in-depth research on the biological function of BTG2 gene in buffalo. 【Method】 The heart,liver,spleen,lung,kidney,small intestine,rumen,uterine horn,mammary gland and ovarian tissues of buffalo were collected.Total RNA was extracted and cDNA was obtained by reverse transcription.The CDS region of BTG2 gene was cloned using buffalo ovary cDNA as template and sequenced.The sequence of BTG2 gene of buffalo and different species were compared and phylogenetic tree was constructed.The physicochemical properties,signal peptides,transmembrane structure,etc.of BTG2 protein were analyzed and predicted by online software.The OE-BTG2 overexpression vector was constructed to transfect buffalo granulosa cells,and the expression of BTG2 gene was detected after transfection.The expression of BTG2 gene in different tissues of buffalo was detected by Real-time quantitative PCR. 【Result】 The CDS region of BTG2 gene in buffalo was 453 bp in length,encoding 150 amino acids.The similarity between buffalo and Bos mutus,Bos taurus,Capra hircus,and Ovis aries exceeded 98%,with the highest similarity (99.8%) observed between buffalo and Bos mutus and Bos taurus.The phylogenetic tree outcomes demonstrated that buffalo had the closest genetic relationship with Bos mutus and Bos taurus,and had the farthest genetic relationship with Mus musculus.BTG2 protein was a hydrophilic protein,which had a btg1 domain at positions 1-108 of the amino acid sequence.A total of 15 phosphorylation sites and 1 O-glycoylation site were observed.It had no signal peptide or transmembrane structure,mainly distributed in the nucleus.BTG2 protein mainly interacted with CNOT7,CNOT8,CNOT11,CNOT9,CNOT1,CNOT10,ATF3,ANKRD49,HES6 and PRMT1 proteins.The secondary structure of BTG2 protein in buffalo was dominated by alpha helix,extended chain and random coils,the prediction result of the tertiary structure was consistent with it.The OE-BTG2 overexpression vector was successfully constructed and transfected into buffalo granulosa cells.Compared with control group (pcDNA3.1(+)),the expression of BTG2 gene in overexpression group (OE-BTG2) was extremely significantly increased (P＜0.01).Real-time quantitative PCR results revealed that BTG2 gene was expressed in heart,liver,spleen,lung,kidney,small intestine,rumen,uterine horn,mammary gland and ovarian tissues of buffalo,with a higher expression in liver than that in other tissues (P＜0.05). 【Conclusion】 BTG2 gene exhibited high conservation among different species,and BTG2 gene was widely expressed in different tissues of buffalo,with the highest expression in liver.The results provided a theoretical basis for further study on the function of BTG2 gene in buffalo.

Key words: buffalo; BTG2 gene; bioinformatics; overexpression vector; tissue expression

中图分类号:

S823.8⁺3

徐媛媛, 谢莹雪, 陆杏蓉, 冯超, 尚江华. 水牛BTG2基因序列分析、过表达载体构建及组织表达研究[J]. 中国畜牧兽医, 2025, 52(9): 4009-4020.

XU Yuanyuan, XIE Yingxue, LU Xingrong, FENG Chao, SHANG Jianghua. Sequence Analysis, Overexpression Vector Construction and Tissue Expression of BTG2 Gene in Buffalo[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4009-4020.

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