2023, 38(2): 275-283. doi: 10.16535/j.cnki.dlhyxb.2022-191
关键词： 尼罗罗非鱼 , Tmbim 6 , 亚细胞定位 , NF-κB , 基因表达
Keywords： Oreochromis niloticus , Tmbim 6 , subcellular localization analysis , NF-κB , expression analysis
为了解跨膜Bax抑制剂母体6(transmembrane Bax inhibitor motif containing 6, TMBIM 6)在尼罗罗非鱼(Oreochromis niloticus)病原体感染中的作用,利用聚合酶链式反应(PCR)对Tmbim 6基因进行克隆与鉴定,并采用实时荧光定量PCR(qPCR)分析Tmbim 6在健康尼罗罗非鱼(体质量为80~100 g)的组织分布模式及无乳链球菌(Streptococcus agalactiae)、Poly I:C模拟病毒刺激后的表达变化。结果表明:尼罗罗非鱼Tmbim 6开放阅读框为714 bp,可编码237个氨基酸,包含6个跨膜结构域和1个C-末端基序;亚细胞定位显示,尼罗罗非鱼TMBIM 6定位于细胞质,双荧光素酶报告基因系统检测发现,Tmbim 6在HEK-293T细胞中过表达后极显著抑制NF-κB通路(P<0.01);qPCR分析显示,Tmbim 6基因在所检测的组织中广泛分布,在肝脏和肌肉中的表达量最高;经无乳链球菌感染和Poly I:C刺激后,Tmbim 6在肝脏、脾脏、头肾、脑和肠道中的表达水平均显著上调(P<0.05)。研究表明,尼罗罗非鱼TMBIM 6参与了无乳链球菌感染和Poly I:C刺激后的细胞应激过程及免疫反应。
为了解跨膜Bax抑制剂母体6(transmembrane Bax inhibitor motif containing 6, TMBIM 6)在尼罗罗非鱼(Oreochromis niloticus)病原体感染中的作用,利用聚合酶链式反应(PCR)对Tmbim 6基因进行克隆与鉴定,并采用实时荧光定量PCR(qPCR)分析Tmbim 6在健康尼罗罗非鱼(体质量为80~100 g)的组织分布模式及无乳链球菌(Streptococcus agalactiae)、Poly I:C模拟病毒刺激后的表达变化。结果表明:尼罗罗非鱼Tmbim 6开放阅读框为714 bp,可编码237个氨基酸,包含6个跨膜结构域和1个C-末端基序;亚细胞定位显示,尼罗罗非鱼TMBIM 6定位于细胞质,双荧光素酶报告基因系统检测发现,Tmbim 6在HEK-293T细胞中过表达后极显著抑制NF-κB通路(P<0.01);qPCR分析显示,Tmbim 6基因在所检测的组织中广泛分布,在肝脏和肌肉中的表达量最高;经无乳链球菌感染和Poly I:C刺激后,Tmbim 6在肝脏、脾脏、头肾、脑和肠道中的表达水平均显著上调(P<0.05)。研究表明,尼罗罗非鱼TMBIM 6参与了无乳链球菌感染和Poly I:C刺激后的细胞应激过程及免疫反应。
| [1] | HVCKELHOVEN R.BAX Inhibitor-1,an ancient cell death suppressor in animals and plants with prokaryotic relatives[J].Apoptosis,2004,9(3):299-307. |
| [2] | WATANABE N,LAM E.Arabidopsis Bax inhibitor-1 functions as an attenuator of biotic and abiotic types of cell death[J].The Plant Journal:for Cell and Molecular Biology,2006,45(6):884-894. |
| [3] | DANIAL N N,KORSMEYER S J.Cell death:critical control points[J].Cell,2004,116(2):205-219. |
| [4] | XU Q,REED J C.Bax inhibitor-1,a mammalian apoptosis suppressor identified by functional screening in yeast[J].Molecular Cell,1998,1(3):337-346. |
| [5] | CARRARA G,SARAIVA N,GUBSER C,et al.Six-transmembrane topology for Golgi anti-apoptotic protein (GAAP) and Bax inhibitor 1 (BI-1) provides model for the transmembrane Bax inhibitor-containing motif (TMBIM) family[J].The Journal of Biological Chemistry,2012,287(19):15896-15905. |
| [6] | CHAE H J,KIM H R,XU C Y,et al.BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress[J].Molecular Cell,2004,15(3):355-366. |
| [7] | ROJAS-RIVERA D,HETZ C.TMBIM protein family:ancestral regulators of cell death[J].Oncogene,2015,34(3):269-280. |
| [8] | LISAK D A,SCHACHT T,ENDERS V,et al.The transmembrane Bax inhibitor motif (TMBIM) containing protein family:tissue expression,intracellular localization and effects on the ER Ca2+-filling state[J].Biochimica et Biophysica Acta,2015,1853(9):2104-2114. |
| [9] | KIM H R,LEE G H,CHO E Y,et al.Bax inhibitor 1 regulates ER-stress-induced ROS accumulation through the regulation of cytochrome P450 2E1[J].Journal of Cell Science,2009,122(Pt 8):1126-1133. |
| [10] | LISAK D,SCHACHT T,GAWLITZA A,et al.BAX inhibitor-1 is a Ca2+ channel critically important for immune cell function and survival[J].Cell Death and Differentiation,2016,23(2):358-368. |
| [11] | JEON K,LIM H,KIM J H,et al.Bax inhibitor-1 enhances survival and neuronal differentiation of embryonic stem cells via differential regulation of mitogen-activated protein kinases activities[J].Biochimica et Biophysica Acta,2012,1823(12):2190-2200. |
| [12] | KITAMURA M.Biphasic,bidirectional regulation of NF-kappaB by endoplasmic reticulum stress[J].Antioxidants & Redox Signaling,2009,11(9):2353-2364. |
| [13] | HENKE N,LISAK D A,SCHNEIDER L,et al.The ancient cell death suppressor BAX inhibitor-1[J].Cell Calcium,2011,50(3):251-260. |
| [14] | CAI J,WEI S N,LU Y S,et al.Bax inhibitor-1 from orange spotted grouper,Epinephelus coioides involved in viral infection[J].Fish & Shellfish Immunology,2018,78:91-99. |
| [15] | CHENG C H,LUO S W,WANG A L,et al.Molecular and immune response characterizations of a novel Bax inhibitor-1 gene in pufferfish,Takifugu obscurus[J].Fish Physiology and Biochemistry,2017,43(4):965-975. |
| [16] | DU Z Q,LAN J F,WENG Y D,et al.BAX inhibitor-1 silencing suppresses white spot syndrome virus replication in red swamp crayfish,Procambarus clarkii[J].Fish & Shellfish Immunology,2013,35(1):46-53. |
| [17] | 苗国英,亓海刚,李莉,等.栉孔扇贝BI-1基因的克隆与表达分析[J].海洋与湖沼,2014,45(6):1251-1257. MIAO G Y,QI H G,LI L,et al.Cloning and expression of bax inhibitor-1 gene of Zhikong scallop Chlamys farreri[J].Oceanologia et Limnologia Sinica,2014,45(6):1251-1257.(in Chinese) |
| [18] | 严楚,彭凯,张志妮,罗非鱼链球菌综合防控技术研究进展[J].广东畜牧兽医科技,2021,46(6):13-16. YAN C,PENG K,ZHANG Z N.Research progress on comprehensive control technology of tilapia Streptococcus[J].Guangdong Journal of Animal and Veterinary Science,2021,46(6):13-16.(in Chinese) |
| [19] | 雷燕,肖洋,赵振峰,等.罗非鱼罗湖病毒RT-PCR检测方法的建立及初步应用[J].广东海洋大学学报,2019,39(3):1-5. LEI Y,XIAO Y,ZHAO Z F,et al.Development and application of a RT-PCR assay of tilapia lake virus[J].Journal of Guangdong Ocean University,2019,39(3):1-5.(in Chinese) |
| [20] | 罗国玲,王孝谦,简纪常,等.尼罗罗非鱼CD209基因的克隆、表达及功能鉴定[J].大连海洋大学学报,2021,36(3):374-382. LUO G L,WANG X Q,JIAN J C,et al.Cloning,expression and functional identification of CD209 gene in Nile tilapia Oreochromis niloticus[J].Journal of Dalian Ocean University,2021,36(3):374-382.(in Chinese) |
| [21] | BULTYNCK G,KIVILUOTO S,HENKE N,et al.The C Terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore[J].The Journal of Biological Chemistry,2012,287(4):2544-2557. |
| [22] | LISBONA F,ROJAS-RIVERA D,THIELEN P,et al.BAX inhibitor-1 is a negative regulator of the ER stress sensor IRE1 alpha[J].Molecular Cell,2009,33(6):679-691. |
| [23] | BAICHWAL V R,BAEUERLE P A.Apoptosis:activate NF-κB or die?[J].Current Biology,1997,7(2):R94-R96. |
| [24] | BEG A A,SHA W C,BRONSON R T,et al.Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-κB[J].Nature,1995,376(6536):167-170. |
| [25] | 罗智文,董志祥,林连兵,等.鱼类重要免疫器官抗菌机制的研究进展[J].水产科学,2021,40(4):624-634. LUO Z W,DONG Z X,LIN L B,et al.Advances on immunological mechanisms of important immune organs against pathogenic microorganisms in fish:a review[J].Fisheries Science,2021,40(4):624-634.(in Chinese) |
| [26] | YU Y Y,WANG Q C,HUANG Z Y,et al.Immunoglobulins,mucosal immunity and vaccination in teleost fish[J].Frontiers in Immunology,2020,11:567941. |
| [27] | LIESCHKE G J,TREDE N S. Fish immunology[J].Current Biology:CB,2009,19(16):R678-R682. |
| [28] | PRETTO-GIORDANO L G,MVLLER E E,DE FREITAS J C,et al.Evaluation on the pathogenesis of Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)[J].Brazilian Archives of Biology and Technology,2010,53(1):87-92. |
| [29] | 胡惠玲,汪志文,黎源,等.尼罗罗非鱼清道夫受体基因Scara3的克隆及病原微生物刺激后的表达响应[J].大连海洋大学学报,2022,37(1):71-79. HU H L,WANG Z W,LI Y,et al.Cloning and expression response of Scara3 gene to pathogenic microorganism stimulation in Nile tilapia Oreochromis niloticus[J].Journal of Dalian Ocean University,2022,37(1):71-79.(in Chinese) |
| [30] | SCHUCHAT A.Epidemiology of group B streptococcal disease in the United States:shifting paradigms[J].Clinical Microbiology Reviews,1998,11(3):497-513. |
| [31] | DANEMAN R,PRAT A.The blood-brain barrier[J].Cold Spring Harbor Perspectives in Biology,2015,7(1):a020412. |
| [32] | URRA H,DUFEY E,LISBONA F,et al.When ER stress reaches a dead end[J].Biochimica et Biophysica Acta,2013,1833(12):3507-3517. |
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