藏鸡GPX3基因的克隆、组织表达谱研究及功能预测

doi:10.13560/j.cnki.biotech.bull.1985.2022-0967

摘要/Abstract

摘要：

本研究对藏鸡GPX3基因进行克隆和生物信息学分析，检测其在藏鸡不同组织中的表达量，鉴定GPX3互作蛋白的mRNA表达水平并分析其相关性，为进一步探究GPX3基因对藏鸡免疫功能的影响奠定基础。本试验以70日龄健康藏鸡（公鸡）作为研究对象，利用RT-PCR技术获得GPX3基因CDS序列并进行生物信息学分析；利用qPCR技术检测GPX3基因在藏鸡心、肝、脾、肺和肾组织中的表达差异情况以及分析可能与GPX3蛋白存在互作关系的10个相关蛋白在脾中的mRNA表达情况及其相关性。结果显示，成功扩增藏鸡GPX3基因799 bp，包括5' UTR 78 bp，CDS区660 bp，3' UTR 61 bp，可编码219个氨基酸。GPX3 mRNA在藏鸡5个组织中均有表达，且在脾中表达量最高，预测GPX3基因在脾中表达量最高这一结果可能与该蛋白的免疫功能有关。GPX3蛋白与预测互作蛋白SOD2呈极显著正相关，推测GPX3在耐药性、生殖调控等方面存在一定的调节作用。本研究成功克隆藏鸡GPX3基因CDS区660 bp，预测其可能与SOD2呈极显著正相关。为进一步了解GPX3基因的抗氧化功能在藏鸡免疫机制中的作用提供理论依据。

关键词: 藏鸡, GPX3, 克隆, 组织表达谱, 功能预测

Abstract:

The aim is to clone and have bioinformatics analysis of GPX3 gene in Tibetan chickens, to detect its expressions in different tissues, and to identify the mRNA expressions of GPX3 interacting proteins and analyse their correlations for laying the foundation for further exploring the effect of GPX3 gene on immune function in Tibetan chickens. In this experiment, having 70-day-old healthy Tibetan chickens as study material, RT-PCR was applied to clone the CDS sequence of GPX3 gene and to perform the biological analysis. Real-time fluorescent quantitative PCR（qPCR）was used to detect the expression differences of GPX3 gene in the hearts, livers, spleens, lungs and kidneys of Tibetan chickens, and to analyze the mRNA expressions of 10 related proteins that may interact with GPX3 protein in spleen. A full length of 799 bp GPX3 gene sequence was cloned successfully, including 78 bp of 5' UTR, 660 bp of CDS, and 61 bp of 3' UTR, encoding 219 amino acids. GPX3 mRNA was expressed in 5 tissues of Tibetan chickens, and the highest in the spleen, predicting that the result of the highest expression of GPX3 in the spleen might be related to this protein immune function. GPX3 protein showed a highly and significantly positive correlation with the predicted reciprocal protein SOD2, suggesting that GPX3 might play an important role in drug resistance and reproductive regulation. A 660 bp CDS of GPX3 gene was cloned successfully in this study, predicting that it might be highly significantly and positively correlated with SOD2. This study may provide a theoretical basis for further understanding the role of the antioxidant function of GPX3 gene in the immune mechanism of Tibetan chickens.

Key words: Tibetan chicken, GPX3, clone, tissue expression profile, function prediction

陈楚雯, 李洁, 赵瑞鹏, 刘媛, 吴锦波, 李志雄. 藏鸡GPX3基因的克隆、组织表达谱研究及功能预测[J]. 生物技术通报, 2023, 39(3): 311-320.

CHEN Chu-wen, LI Jie, ZHAO Rui-peng, LIU Yuan, WU Jin-bo, LI Zhi-xiong. Cloning, Tissue Expression Profile and Function Prediction of GPX3 Gene in Tibetan Chicken[J]. Biotechnology Bulletin, 2023, 39(3): 311-320.

图/表 10

表1 软件用途

Table 1 Software usage

软件Software brand	用途Usage
ORF Finder	预测开放阅读框
DNAMAN	核苷酸序列翻译
MEGA 5.0	构建系统进化树
Clustalx 2.1	多序列比对
MegAlign	同源性分析
ExPASy ProtScale	疏水性预测
TMHMM-2.0	蛋白质跨膜区分析
SignalP-4.1	信号肽预测
SOPMA	蛋白质二级结构预测
SWISS-MODEL	蛋白质三级结构预测
PSORT II	蛋白质亚细胞定位预测
Net Phos 3.1	蛋白质磷酸化位点预测
STRING	互作蛋白预测

表2 引物信息

Table 2 Primer information

目的基因Gene	引物序列Primer sequence（5'-3'）	登录号Accession No.	退火温度Annealing temperature T_m/℃
GSR	S: GCAAGGAGGAGAAGGTGGTG	XM_015276627.4	60
	A: TTGTCAAAGTCGGCCTTGGT
CAT	S: GCTGAAGCTGGGAAAAAGGATG	NM_001031215.2	60
	A: TCCTGCAGTTGTATGGACGC
SOD2	S: GCCACCTACGTGAACAACCT	NM_204211.2	60
	A: ACCTGAGCTGTAACATCACCTTT
SOD1	S: CAAATGGGTGTACCAGCGCA	NM_205064.2	60
	A: CAAATGGGTGTACCAGCGCA
HPGDS	S: CTGCACCCAAGGACCCAT	NM_205011.2	60
	A: GTCTGCTCCTTCCAACCTGT
GSTA3	S: CGTCGTCCAACCAGCAGATA	NM_001001777.2	60
	A: CCGTGGTCCTTCAAAACCTTC
GSTA	S: GATCTCCCACAGCCGACAC	XM_046938585.1	60
	A: AGCTCTCCTTTCCAGAGGGC
GSS	S: TTGCTGGGCTGTACTCACTG	XM_025142428.3	60
	A: ACAGGTTGTTCCCTCCTCCT
SOD3	S: TTGTGTCCGATCCCACCTCG	XM_040699307.2	60
	A: CTGGTGAGTGAGAACCTGCCC
PRDX6	S: CCTGTGGACTGGAAGTGTGG	NM_001039329.3	60
	A: ACGCAGGTACTTCTTGCCTG
GAPDH	S: CTGCCCAGAACATCATCCCA	NM_204305.2	60
	A: CGGCAGGTCAGGTCAACAAC

图1 PCR扩增产物电泳图

Fig. 1 Electrophoresis of PCR amplified products

图2 藏鸡GPX3基因CDS区核苷酸及预测氨基酸序列

Fig. 2 Nucleotide and predicted amino acid sequence of CDS region of GPX3 gene in Tibetan chicken

图3 藏鸡与原鸡GPX3蛋白氨基酸序列对比

Fig. 3 Comparison of amino acid sequences of GPX3 prot-ein between Tibetan chicken and native chicken

图4 藏鸡GPX3基因的生物信息学分析 A. 藏鸡GPX3基因同源性分析; B. GPX3基因进化树分析

Fig. 4 Bioinformatics analysis of Tibetan chicken GPX3 gene A. Homology analysis of GPX3 gene in Tibetan chicken. B. Evolutionary tree analysis of GPX3 gene

图5 藏鸡GPX3蛋白的生物信息学分析 A. 藏鸡GPX3蛋白的疏水性分析；B. 藏鸡GPX3蛋白的跨膜区分析；C. 藏鸡GPX3蛋白的信号肽预测；D. 藏鸡GPX3蛋白的磷酸化位点预测

Fig. 5 Bioinformatics analysis of Tibetan chicken GPX3 protein A. Hydrophobicity analysis of Tibetan chicken GPX3 protein. B. Transmembrane region analysis of Tibetan chicken GPX3 protein. C. Predicted signal peptide of Tibetan chicken GPX3 protein. D. Predicted phosphorylation site of Tibetan chicken GPX3 protein

图6 藏鸡GPX3蛋白的生物信息学分析 A. 藏鸡GPX3蛋白二级结构预测；B. 藏鸡GPX3蛋白三级结构预测；C. 与GPX3蛋白存在相互作用的蛋白预测

Fig. 6 Bioinformatics analysis of Tibetan chicken GPX3 protein A. Prediction of the secondary structure of GPX3 protein in Tibetan chicken. B. Prediction of the tertiary structure of GPX3 protein in Tibetan chicken. C. Prediction of proteins interacting with GPX3 protein

图7 藏鸡GPX3基因在藏鸡不同组织中的相对表达量不同大写字母代表差异极显著（P < 0.01）

Fig. 7 Relative expression of GPX3 gene in different tissues of Tibetan chicken Different capital letters mean significant difference（P < 0.01）

表3 GPX3互作蛋白基因表达量之间的相关性分析

Table 3 Correlation analysis of genes expression among proteins interacting with GPX3

基因 Gene	GPX3	GSR	CAT	SOD2	SOD1	HPGDS	GSTA3	GSTA	GSS	SOD3	PRDX6
GPX3	1
GSR	-0.330	1
CAT	0.383	-0.375	1
SOD2	0.773**	-0.066	-0.205	1
SOD1	0.401	-0.297	0.978**	-0.209	1
HPGDS	-0.325	0.992**	-0.379	-0.062	-0.296	1
GSTA3	-0.253	0.297	-0.443	0.186	-0.481	0.289	1
GSTA	-0.344	-0.440	0.312	-0.465	0.241	-0.455	0.387	1
GSS	-0.266	-0.540*	0.348	-0.605*	0.327	-0.549*	-0.708**	0.237	1
SOD3	-0.156	-0.276	-0.226	-0.194	-0.226	-0.279	-0.693**	-0.363	0.696**	1
PRDX6	0.428	0.054	-0.631*	0.871**	-0.644**	0.055	0.341	-0.491	-0.599*	0.005	1

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