海南黑山羊MBL2基因的转录调控元件的筛选与分析

doi:10.11843/j.issn.0366-6964.2022.06.013

摘要/Abstract

摘要： 甘露聚糖结合凝集素C(mannose binding lectin C，MBL-C)是C型(Ca²⁺依赖型)凝集素超家族的成员，其作为一种急性期蛋白，具有抗细菌感染的功能，参与机体的天然免疫反应。为鉴定出结合在MBL2基因启动子区(1 009 bp)的重要转录因子，探寻该基因的转录调控机制，本研究选取海南黑山羊MBL2基因的启动子序列1 009 bp，采用DNA重组技术克隆6个转录起始位点上游1 009 bp的启动子5'端侧翼缺失序列，克隆片段经双酶切后连接至pGL3-Basic载体。重组质粒转染至293T细胞中，结合双荧光素酶活性检测系统筛选MBL2基因的核心启动子区域。通过在线生物信息学软件预测山羊MBL2基因的核心启动子区域的转录因子结合位点，利用点突变技术构建转录因子结合位点缺失的载体，转染293T细胞后结合双荧光素酶活性检测系统分析其转录活性。结果表明，海南黑山羊MBL2基因的核心启动子区域位于转录起始位点上游-304~-45 bp范围内，在线软件分析该区域存在RELA、NF-κB2、MZF1等3种转录因子结合位点。双荧光素酶报告分析结果表明，RELA和NF-κB2的结合位点缺失后均使山羊MBL2基因的转录活性极显著下降(P < 0.01)。结果提示，RELA和NF-κB2对山羊MBL2基因的转录活性可能具有重要的正调控作用。该研究为进一步探寻海南黑山羊MBL2基因的功能提供理论依据。

关键词: 海南黑山羊, MBL2基因, 转录调控, 双荧光素酶, 核心启动子

Abstract: Mannose binding lectin C(MBL-C)is a member of the C-type (Ca²⁺dependent) lectin superfamily. As an acute phase protein, MBL-C plays an important role in the innate immune response. The aim of this study was to identify the key transcriptional factors in the promoter region (1 009 bp) of MBL2 gene, and explore the expression regulation mechanism of MBL2 gene. The promoter sequence 1 009 bp of MBL2 gene was selected from Hainan black goat, and a serial of 5'-flanking deletion regions of the goat MBL2 promoter were amplified and subcloned into pGL3-Basic vector. The recombined plasmids were transfected into 293T cells and detected with the dual-luciferase activity detection system to screen the core promoter region of MBL2 gene. The transcription factor binding site of the core promoter region of Hainan black goat MBL2 gene were predicted by online bioinformatics software. The site mutation technology and dual-luciferase activity detection system were utilized to identify the transcription factor binding sites. The results showed that the core promoter region of Hainan black goat MBL2 gene located in the range of -304 to -45 bp of the transcription initiation site. Online software analysis indicated that this region existed three transcription factors binding sites, including RELA, NF-κB2 and MZF1. Dual-luciferase report analysis results showed that the deletion of the binding sites of RELA and NF-κB2 extremely significantly reduced the transcriptional activity of goat MBL2 gene (P < 0.01). These results indicated that RELA and NF-κB2 might positively activate transcriptional regulation of goat MBL2 gene. This study can provide a theoretical basis for further exploring the function of MBL2 gene in Hainan black goat.

Key words: Hainan black goat, MBL2 gene, transcriptional regulation, dual-luciferase, core promoter

中图分类号:

S827.2

王雪梅, 翟哲, 陈巧玲, 吴艳茹, 吴昊天, 黄惠娴, 刘志勇, 李崇瑞, 满初日嘎, 王凤阳, 杜丽, 陈思. 海南黑山羊MBL2基因的转录调控元件的筛选与分析[J]. 畜牧兽医学报, 2022, 53(6): 1795-1806.

WNAG Xuemei, ZHAI Zhe, CHEN Qiaoling, WU Yanru, WU Haotian, HUANG Huixian, LIU Zhiyong, LI Chongrui, MANCHU Riga, WANG Fengyang, DU Li, CHEN Si. Screening and Analysis of Transcriptional Regulatory Elements of MBL2 Gene in Hainan Black Goat[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1795-1806.

参考文献 34

[1]	ZHOU H H, ZHENG X L, MA T M, et al. Genotype identification and phylogenetic analysis of Enterocytozoon bieneusi in farmed black goats (Capra hircus) from China's Hainan Province[J]. Parasite, 2019, 26(7): 62.
[2]	YE Y X, LEI X L, CHENG W K, et al. Research progress and some thoughts of industrial development of Hainan Black Goats[J]. Chinese Journal of Tropical Agriculture, 2016, 36(10): 114-118. (in Chinese)叶玉秀, 雷湘兰, 程文科, 等. 海南黑山羊研究进展与产业发展思路[J]. 热带农业科学, 2016, 36(10): 114-118.
[3]	ZHANG K Z, TAO P, LIU J X, et al. Distinct expression profile and histological distribution of NLRP3 inflammasome components in the tissues of Hainan black goat suggest a site-specific role in the inflammatory response[J]. Acta Vet Hung, 2017, 65(3): 402-416.
[4]	IDOWU P A, IDOWU A P, ZISHIRI O T, et al. Activity of mannose-binding lectin on bacterial-infected chickens-A review[J]. Animals(Basel), 2021, 11(3): 787.
[5]	KALIA N, SINGH J, KAUR M. The ambiguous role of mannose-binding lectin (MBL) in human immunity[J]. Open Med (Wars), 2021, 16(1): 299-310.
[6]	CHEN Y, HU M, DENG F, et al. Mannan-binding lectin deficiency augments hepatic endoplasmic reticulum stress through IP3R-controlled calcium release[J]. Cell Calcium, 2021, 100: 102477.
[7]	GADJEVA M, TAKAHASHI K, THIEL S. Mannan-binding lectin-a soluble pattern recognition molecule[J]. Mol Immunol, 2004, 41(2-3): 113-121.
[8]	ZHENG M F, SHI S Y, WEI W, et al. Correlation between MBL2/CD14/TNF-α gene polymorphisms and susceptibility to spinal tuberculosis in Chinese population[J]. Biosci Rep, 2018, 38(1): BSR20171140.
[9]	MEDETALIBEYOGLU A, BAHAT G, SENKAL N, et al. Mannose binding lectin gene 2 (rs1800450) missense variant may contribute to development and severity of COVID-19 infection[J]. Infect Genet Evol, 2021, 89: 104717.
[10]	MORETTI R, SOGLIA D, CHESSA S, et al. Identification of SNPs associated with somatic cell score in candidate genes in Italian Holstein Friesian Bulls[J]. Animals (Basel), 2021, 11(2): 366.
[11]	GIANG N T, VAN TONG H, QUYET D, et al. Complement protein levels and MBL2 polymorphisms are associated with dengue and disease severity[J]. Sci Rep, 2020, 10(1): 14923.
[12]	FRASER R S, LUMSDEN J S, LILLIE B N. Identification of polymorphisms in the bovine collagenous lectins and their association with infectious diseases in cattle[J]. Immunogenetics, 2018, 70(8): 533-546.
[13]	WEN D X, CHEN J C, WANG Y F, et al. Screening of SNPs related to antibacterial activity in the 3'-UTR region in MBL2 gene of yak[J]. Chinese Veterinary Science, 2021, 51(7): 898-907. (in Chinese)温东旭, 陈建春, 王一飞, 等. 牦牛MBL2基因3'-UTR区抗菌相关SNPs的筛选[J]. 中国兽医科学, 2021, 51(7): 898-907.
[14]	CAPPARELLI R, PARLATO M, AMOROSO M G, et al. Mannose-binding lectin haplotypes influence Brucella abortus infection in the water buffalo (Bubalus bubalis)[J]. Immunogenetics, 2008, 60(3-4): 157-165.
[15]	YUAN Z C, XU W D, LAN Y Y, et al. Association of MBL2 gene polymorphisms and systemic lupus erythematosus susceptibility: a meta-analysis[J]. Int J Rheum Dis, 2021, 24(2): 147-158.
[16]	BROWN G D, WILLMENT J A, WHITEHEAD L. C-type lectins in immunity and homeostasis[J]. Nat Rev Immunol, 2018, 18(6): 374-389.
[17]	BOSCHMANN S E, GOELDNER I, TUON F F, et al. Mannose-binding lectin polymorphisms and rheumatoid arthritis: a short review and meta-analysis[J]. Mol Immunol, 2016, 69: 77-85.
[18]	WEN D X, ZHANG P Y, XU Y F, et al. Expression analyses of yak MBL2 gene mRNA and related miRNAs in different tissues[J]. Chinese Veterinary Science, 2020, 50(7): 886-897. (in Chinese)温东旭, 张鹏宇, 徐业芬, 等. 牦牛MBL2基因mRNA及相关miRNAs在不同组织中的表达分析[J]. 中国兽医科学, 2020, 50(7): 886-897.
[19]	TIYO B T, VENDRAMINI E C L, DE SOUZA V H, et al. Association of MBL2 Exon 1 polymorphisms with multibacillary leprosy[J]. Front Immunol, 2020, 11: 1927.
[20]	ZHAO Z L. The study of MBL2 gene and dairy cattle mastitis resistance[J]. Changchun: Jinlin University, 2012: 1-141. (in Chinese)赵中利. MBL2基因与奶牛乳腺炎抗性研究[D]. 长春: 吉林大学, 2012: 1-141.
[21]	VASILYEV S A, TOLMACHEVA E N, VASILYEVA O Y, et al. LINE-1 retrotransposon methylation in chorionic villi of first trimester miscarriages with aneuploidy[J]. J Assist Reprod Genet, 2021, 38(1): 139-149.
[22]	URBAN M B, BAEUERLE P A. The 65-kD subunit of NF-kappa B is a receptor for I kappa B and a modulator of DNA-binding specificity[J]. Genes Dev, 1990, 4(11): 1975-1984.
[23]	DUCKETT C S, PERKINS N D, KOWALIK T F, et al. Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3)[J]. Mol Cell Biol, 1993, 13(3): 1315-1322.
[24]	YANG Y. NF-κB target genes spectrum and study of gene regulatory networks[D]. Nanjing: Southeast University, 2016: 1-149. (in Chinese)杨洋. NF-κB靶基因谱及其调控网络的研究[D]. 南京: 东南大学, 2016: 1-149.
[25]	CHAWLA M, MUKHERJEE T, DEKA A, et al. An epithelial Nfkb2 pathway exacerbates intestinal inflammation by supplementing latent RelA dimers to the canonical NF-κB module[J]. Proc Natl Acad Sci U S A, 2021, 118(25): e2024828118.
[26]	CAO J P, WANG H J, YU J K, et al. The involvement of NF-κB p65/p52 in the effects of GDNF on DA neurons in early PD rats[J]. Brain Res Bull, 2008, 76(5): 505-511.
[27]	CAO J P, NIU H Y, WANG H J, et al. NF-κB p65/p52 plays a role in GDNF up-regulating Bcl-2 and Bcl-w expression in 6-OHDA-induced apoptosis of MN9D cell[J]. Int J Neurosci, 2013, 123(10): 705-710.
[28]	NGO K A, KISHIMOTO K, DAVIS-TURAK J, et al. Dissecting the regulatory strategies of NF-κB RelA target genes in the inflammatory response reveals differential transactivation logics[J]. Cell Rep, 2020, 30(8): 2758-2775.e6.
[29]	CHEN S, JIANG S C, ZHENG W, et al. RelA/p65 inhibition prevents tendon adhesion by modulating inflammation, cell proliferation, and apoptosis[J]. Cell Death Dis, 2017, 8(3): e2710.
[30]	KIM Y, ALLEN E, BAIRD L A, et al. NF-κB RELA is required for hepatoprotection during pneumonia and sepsis[J]. Infect Immun, 2019, 87(8): e00132-19.
[31]	ZHANG J B, YIN S H, LU A F. The role of NF-κb signaling pathway in the infection of mycoplasma pneumoniae in sheep alveolar epithelial cell[J]. Journal of Inner Mongolia University (Natural Science Edition), 2018, 49(3): 296-303. (in Chinese)张俊波, 印双红, 陆安法. NF-κB信号通路调控绵羊肺炎支原体感染宿主肺泡上皮细胞的作用机理[J]. 内蒙古大学学报: 自然科学版, 2018, 49(3): 296-303.
[32]	JOBE F, SIMPSON J, HAWES P, et al. Respiratory syncytial virus sequesters NF-κB subunit p65 to cytoplasmic inclusion bodies to inhibit innate immune signaling[J]. J Virol, 2020, 94(22): e01380-20.
[33]	LOUGARIS V, TABELLINI G, VITALI M, et al. Defective natural killer-cell cytotoxic activity in NFKB2-mutated CVID-like disease[J]. J Allergy Clin Immunol, 2015, 135(6): 1641-1643.e3.
[34]	GUO G Q, YE S S, XIE S D, et al. The cytomegalovirus protein US31 induces inflammation through mono-macrophages in systemic lupus erythematosus by promoting NF-κB2 activation[J]. Cell Death Dis, 2018, 9(2): 104.