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

Abstract

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

CLC Number:

S823.8⁺3

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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Sequence Analysis, Overexpression Vector Construction and Tissue Expression of BTG2 Gene in Buffalo

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