Cloning, Tissue Expression Profile and Function Prediction of GPX3 Gene in Tibetan Chicken

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

Abstract

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

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.

Figures/Tables 10

References 30

[1]	张宏亮. 微量元素硒在畜禽养殖中的研究进展[J]. 饲料博览, 2021(9): 19-21.
	Zhang HL. Research progress of trace element selenium in livestock and poultry breeding[J]. Feed Rev, 2021(9): 19-21.
[2]	Hariharan S, Dharmaraj S. Selenium and selenoproteins: it’s role in regulation of inflammation[J]. Inflammopharmacology, 2020, 28(3): 667-695. doi: 10.1007/s10787-020-00690-x pmid: 32144521
[3]	张丹丹, 娄鹏博, 李振. GPxs家族的研究进展[J]. 农业技术与装备, 2012(15): 66-67.
	Zhang DD, Lou PB, Li Z. Research progress of GPxs family[J]. Agric Technol ＆ Equip, 2012(15): 66-67.
[4]	Kipp AP. Selenium-dependent glutathione peroxidases during tumor development[J]. Adv Cancer Res, 2017, 136: 109-138. doi: S0065-230X(17)30022-2 pmid: 29054415
[5]	Nirgude S, Choudhary B. Insights into the role of GPX3, a highly efficient plasma antioxidant, in cancer[J]. Biochem Pharmacol, 2021, 184: 114365. doi: 10.1016/j.bcp.2020.114365 URL
[6]	Avissar N, Ornt DB, Yagil Y, et al. Human kidney proximal tubules are the main source of plasma glutathione peroxidase[J]. Am J Physiol, 1994, 266(2 Pt 1):C367-C375. doi: 10.1152/ajpcell.1994.266.2.C367 URL
[7]	Khan AQ, Rashid K, AlAmodi AA, et al. Reactive oxygen species(ROS)in cancer pathogenesis and therapy: an update on the role of ROS in anticancer action of benzophenanthridine alkaloids[J]. Biomed Pharmacother, 2021, 143: 112142. doi: 10.1016/j.biopha.2021.112142 URL
[8]	Yi ZH, Jiang L, Zhao L, et al. Glutathione peroxidase 3(GPX3)suppresses the growth of melanoma cells through reactive oxygen species(ROS)-dependent stabilization of hypoxia-inducible factor 1-α and 2-α[J]. J Cell Biochem, 2019, 120(11): 19124-19136. doi: 10.1002/jcb.v120.11 URL
[9]	宋敏, 李咏, 王亚楠, 等. GPX3 DNA甲基化在恶性肿瘤发生发展中作用的研究进展[J]. 现代肿瘤医学, 2022, 30(1): 167-171.
	Song M, Li Y, Wang YN, et al. Research progress on the role of GPX3 DNA methylation in the development and progression of malignant tumors[J]. J Mod Oncol, 2022, 30(1): 167-171.
[10]	Jacobson MD. Reactive oxygen species and programmed cell death[J]. Trends Biochem Sci, 1996, 21(3): 83-86. pmid: 8882579
[11]	国家畜禽遗传资源委员会组. 中国畜禽遗传资源志-家禽志[M]. 北京: 中国农业出版社, 2011.
	China National Commission of Animal Genetic Resources. Animal genetic resources in China[M]. Beijing: Chinese Agriculture Press, 2011.
[12]	郭文场, 丁向清, 张亚兰, 等. 中国藏鸡的特性、饲养管理和保种[J]. 特种经济动植物, 2012, 15(11): 7-11.
	Guo WC, Ding XQ, Zhang YL, et al. Characteristics, feeding management and breeding conservation of Chinese Tibetan chicken[J]. Special Econ Animal Plant, 2012, 15(11): 7-11.
[13]	Pérez-Torres I, Zuniga-Munoz AM, Guarner-Lans V. Beneficial effects of the amino acid glycine[J]. Mini Rev Med Chem, 2017, 17(1): 15-32. pmid: 27292783
[14]	Razak MA, Begum PS, Viswanath B, et al. Multifarious beneficial effect of nonessential amino acid, glycine: a review[J]. Oxid Med Cell Longev, 2017, 2017: 1716701.
[15]	付开斌, 陈祥, 周志楠, 等. 黔北麻羊GPx3基因克隆、生物信息学分析及在不同性腺组织中的表达分析[J]. 中国畜牧杂志, 2022, 58(1): 77-84.
	Fu KB, Chen X, Zhou ZN, et al. Cloning, bioinformatics analysis and expression analysis of GPx3 gene in different gonad tissues of Qianbei ma goat[J]. Chin J Animal Sci, 2022, 58(1): 77-84.
[16]	郭笑, 陈冬雪, 韩笑, 等. 人谷胱甘肽过氧化物酶的生物信息学分析[J]. 北华大学学报: 自然科学版, 2016, 17(6): 750-755.
	Guo X, Chen DX, Han X, et al. Bioinformatics analysis of human glutathione peroxidase[J]. J Beihua Univ Nat Sci, 2016, 17(6): 750-755.
[17]	程雪艳, 蒋国萍, 柴雪良, 等. 泥蚶谷胱甘肽过氧化物酶基因的全长克隆与表达分析[J]. 水生生物学报, 2016, 40(6): 1144-1151.
	Cheng XY, Jiang GP, Chai XL, et al. Full-length cDNA cloning and expression analysis of glutathione peroxidase from blood clam Tegillarca granosa[J]. Acta Hydrobiol Sin, 2016, 40(6): 1144-1151.
[18]	Viéitez C, Busby BP, Ochoa D, et al. High-throughput functional characterization of protein phosphorylation sites in yeast[J]. Nat Biotechnol, 2022, 40(3): 382-390. doi: 10.1038/s41587-021-01051-x
[19]	Zhang SL, Duan X. Prediction of protein subcellular localization with oversampling approach and Chou’s general PseAAC[J]. J Theor Biol, 2018, 437: 239-250. doi: 10.1016/j.jtbi.2017.10.030 URL
[20]	Yang YH, Wu WY, Liu T, et al. A robust method for protein depletion based on gene editing[J]. Methods, 2021, 194: 3-11. doi: 10.1016/j.ymeth.2021.03.001 pmid: 33705859
[21]	Khoso PA, Yang ZJ, Liu CP, et al. Selenoproteins and heat shock proteins play important roles in immunosuppression in the bursa of Fabricius of chickens with selenium deficiency[J]. Cell Stress Chaperones, 2015, 20(6): 967-978. doi: 10.1007/s12192-015-0625-9 URL
[22]	Patterson MJ, McKenzie CG, Smith DA, et al. Ybp1 and Gpx3 signaling in Candida albicans govern hydrogen peroxide-induced oxidation of the Cap1 transcription factor and macrophage escape[J]. Antioxid Redox Signal, 2013, 19(18): 2244-2260. doi: 10.1089/ars.2013.5199 URL
[23]	Yang ZJ, Wang SC, Yin K, et al. miR-1696/GPx3 axis is involved in oxidative stress mediated neutrophil extracellular traps inhibition in chicken neutrophils[J]. J Cell Physiol, 2021, 236(5): 3688-3699. doi: 10.1002/jcp.30105 pmid: 33044016
[24]	Zhao L, Feng Y, Deng J, et al. Selenium deficiency aggravates aflatoxin B1-induced immunotoxicity in chick spleen by regulating 6 selenoprotein genes and redox/inflammation/apoptotic signaling[J]. J Nutr, 2019, 149(6): 894-901. doi: 10.1093/jn/nxz019 pmid: 31070734
[25]	Wang W, Jin YF, Zeng NX, et al. SOD2 facilitates the antiviral innate immune response by scavenging reactive oxygen species[J]. Viral Immunol, 2017, 30(8): 582-589. doi: 10.1089/vim.2017.0043 pmid: 28574756
[26]	Alateyah N, Gupta I, Rusyniak RS, et al. SOD2, a potential transcriptional target underpinning CD44-promoted breast cancer progression[J]. Molecules, 2022, 27(3): 811. doi: 10.3390/molecules27030811 URL
[27]	易力, 刘宏雷. 超氧化物歧化酶2与肿瘤关系的研究进展[J]. 山东医药, 2021, 61(29): 109-112.
	Yi L, Liu HL. Progress of research on the relationship between superoxide dismutase 2 and tumors[J]. Shandong Med J, 2021, 61(29): 109-112.
[28]	Scheurmann J, Treiber N, Weber C, et al. Mice with heterozygous deficiency of manganese superoxide dismutase(SOD2)have a skin immune system with features of “inflamm-aging”[J]. Arch Dermatol Res, 2014, 306(2): 143-155. doi: 10.1007/s00403-013-1389-7 URL
[29]	李曼. SOD2通过LncRNA CLCA3P促进肝细胞ABCC2的表达并提高对药物毒性的耐受[D]. 福州: 福建医科大学, 2020.
	Li M. SOD2 promotes the expression of ABCC2 through lncRNA CLCA3p and improves the detoxification capability of liver cells[D]. Fuzhou: Fujian Medical University, 2020.
[30]	Cepas V, González-Menéndez P, Álvarez-Artime A, et al. SOD2 levels alter reproductive physiology in mice[J]. Free Radic Biol Med, 2016, 96: S32.

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

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

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

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

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