草鱼自噬相关基因Beclin1的克隆及其在MC-LR胁迫下的表达特征
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摘要
为评估微囊藻毒素-LR (MC-LR)对鱼类自噬的影响,根据转录组测序结果得到的EST序列,采用RACE技术获得了草鱼(Ctenopharygodon idella)自噬基因Beclin1 (CiBeclin1)的cDNA全长序列。该基因序列全长1590 bp,包括1344 bp的开放阅读框,编码447个氨基酸,分子量为51.2 kD,理论等电点为4.88。CiBeclin1包含1个BH3和1个ECD结构域。CiBeclin1氨基酸序列与其他物种的相似性为88%—98%,构建的进化树显示CiBeclin1与稀有鲫(Gobiocypris rarus)的Beclin1亲缘关系最近。实时荧光定量PCR (qRT-PCR)检测结果表明, CiBeclin1在肝、肾、脾等10种不同组织中均广泛表达,但在肝脏组织中的相对表达量最丰富,显著高于相对表达量最低的头肾组织(P<0.05)。在不同剂量(25和100μg MC-LR/kg BW) MC-LR胁迫24h、48h、72h和96h后,草鱼肝脏中CiBeclin1表达量均显著低于对照组的表达水平,研究结果表明, MC-LR能抑制草鱼CiBeclin1的表达,但MC-LR是否在该剂量下诱导了草鱼的自噬还有待于进一步研究。
To estimate the effects of Microcystin-LR(MC-LR) on the autophagy, we cloned Beclin1 CiBeclin1 gene from grass carp(Ctenopharygodon idella) using rapid amplification of cDNA ends(RACE) based on the EST sequence. The deduced amino acid sequence of CiBeclin1 was comprised of 447 amino acids, and CiBeclin1 possessed a typical Bcl-2 homology domain 3(BH3) and an evolutionarily conserved domain(ECD). The similarity of CiBeclin1 amino acid sequence with other species was 88%—98%, and phylogenetic analysis demonstrated that CiBeclin1 formed a clade with rare minnow Beclin1. CiBeclin1 was ubiquitously expressed in 10 tested tissues, including the liver, kidney, spleen and others, with the highest level in liver. MC-LR significantly decreased the expression of CiBeclin1 at 24,48, 72 and 96 h, indicating that CiBeclin1 gene might play a regulatory role against MC-LR toxicity. However, whether the used doses of MC-LR could induce liver autophagy needs further investigation.
To estimate the effects of Microcystin-LR(MC-LR) on the autophagy, we cloned Beclin1 CiBeclin1 gene from grass carp(Ctenopharygodon idella) using rapid amplification of cDNA ends(RACE) based on the EST sequence. The deduced amino acid sequence of CiBeclin1 was comprised of 447 amino acids, and CiBeclin1 possessed a typical Bcl-2 homology domain 3(BH3) and an evolutionarily conserved domain(ECD). The similarity of CiBeclin1 amino acid sequence with other species was 88%—98%, and phylogenetic analysis demonstrated that CiBeclin1 formed a clade with rare minnow Beclin1. CiBeclin1 was ubiquitously expressed in 10 tested tissues, including the liver, kidney, spleen and others, with the highest level in liver. MC-LR significantly decreased the expression of CiBeclin1 at 24,48, 72 and 96 h, indicating that CiBeclin1 gene might play a regulatory role against MC-LR toxicity. However, whether the used doses of MC-LR could induce liver autophagy needs further investigation.
引文
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[26]Li X P,Chi H,Zhang J.Beclin-1 is involved in Tongue Sole Cynoglossus semilaevis immune defense against bacterial infection[J].Fish and Shellfish Immunology,2018,77:8-12
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[2]Puddick J,Prinsep M R,Wood S A,et al.High levels of structural diversity observed in microcystins from microcystis CAWBG11 and characterization of six new microcystin congeners[J].Marine Drugs,2014,12(11):5372-5395
[3]Chen J,Xie P,Li L,et al.First identification of the hepatotoxic microcystins in the serum of a chronically exposed human population together with indication of hepatocellular damage[J].Toxicololgy Science,2009,108(1):81-89
[4]Magalhaes V F,Soares R M,Azevedo S M.Microcystin contamination in fish from the jacarepagua lagoon(Rio de Janeiro,Brazil):ecological implication and human health risk[J].Toxicon,2001,39(7):1077-1085
[5]Chen L,Xie P.Mechanisms of microcystin-induced cytotoxicity and apoptosis[J].Mini Reviews in Medicinal Chemistry,2016,16(13):1018-1031
[6]MacKintosh C,Beattie K A,Klumpp S,et al.Cyanobacterial microcystin-lr is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants[J].FEBS Letters,1990,264(2):187-192
[7]Steiner K,Hagenbuch B,Dietrich D R.Molecular cloning and functional characterization of a rainbow trout liver Oatp[J].Toxicology and Applied Pharmacology,2014,280(3):534-542
[8]Menezes C,Alverca E,Dias E,et al.Involvement of endoplasmic reticulum and autophagy in microcystin-lr toxicity in Vero-E6 and HepG2 cell lines[J].Toxicology in Vitro,2013,27(1):138-148
[9]Wu Z,Zhang Y,Bei Y C,et al.Exploring the effects of MC-LR on mitochondrial function in mouse liver[J].Progress in Modern Biomedicine,2014,14(2):226-229[吴珍,张亚,贝云成,等.微囊藻毒素-LR对小鼠肝细胞线粒体功能的影响.现代生物医学进展,2014,14(2):226-229]
[10]Scarlatti F,Maffei R,Beau I,et al.Role of non-canonical beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells[J].Cell Death and Differentiation,2008,15(8):1318-1329
[11]Burman C,Ktistakis N T.Autophagosome formation in mammalian cells[J].Seminars in Immunopathology,2010,32(4):397-413
[12]Liang X H,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by Beclin 1[J].Nature,1999,402(6762):672-676
[13]Aita V M,Liang X H,Murty V V,et al.Cloning and genomic organization of beclin 1,a candidate tumor suppressor gene on chromosome 17Q21[J].Genomics,1999,59(1):59-65
[14]Duan K.The functional study of becn1 in zebrafish[D].Thesis for master of Science,Southwest University.2016[段昆.斑马鱼becn1基因的功能研究.硕士学位论文,西南大学.2016]
[15]Kong H J,Moon J Y,Nam B H,et al.Molecular characterization of the autophagy-related gene Beclin-1 from the Olive Flounder(Paralichthys olivaceus)[J].Fish and Shellfish Immunology,2011,31(2):189-195
[16]Gao D,Xu Z,Kuang X,et al.Molecular characterization and expression analysis of the autophagic gene Beclin 1from the Purse Red Common Carp(Cyprinus carpio)exposed to cadmium[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2014,160:15-22
[17]Liu X H,Wang Z J,Chen D M,et al.Molecular characterization of Beclin 1 in Rare Minnow(Gobiocypris rarus)and its expression after waterborne cadmium exposure[J].Fish Physiology and Biochemistry,2016,42(1):111-123
[18]Wei L,Liu Y,Zhong S,et al.Transcriptome analysis of grass carp provides insights into the immune-related genes and pathways in response to mc-lr induction[J].Aquaculture,2018,488:207-216
[19]Kang R,Zeh H J,Lotze M T,et al.The Beclin 1 network regulates autophagy and apoptosis[J].Cell Death and Differentiation,2011,18(4):571-580
[20]Liang X H,Yu J,Brown K,et al.Beclin 1 contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function[J].Cancer Research,2001,61(8):3443-3449
[21]Pattingre S,Tassa A,Qu X,et al.Bcl-2 antiapoptotic proteins inhibit beclin 1-dependent autophagy[J].Cell,2005,122(6):927-939
[22]Furuya N,Yu J,Byfield M,et al.The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding,autophagy and tumor suppressor function[J].Autophagy,2005,1(1):46-52
[23]Lin H X,Qiu H J,Zeng F,et al.Decreased expression of Beclin 1 correlates closely with Bcl-xL expression and poor prognosis of ovarian carcinoma[J].PLoS One,2013,8(4):e60516
[24]Levine B,Kroemer G.Autophagy in the pathogenesis of disease[J].Cell,2008,132(1):27-42
[25]Sahu R,Kaushik S,Clement C C,et al.Microautophagy of cytosolic proteins by late endosomes[J].Developmental Cell,2011,20(1):131-139
[26]Li X P,Chi H,Zhang J.Beclin-1 is involved in Tongue Sole Cynoglossus semilaevis immune defense against bacterial infection[J].Fish and Shellfish Immunology,2018,77:8-12
[27]Kroemer G,Marino G,Levine B.Autophagy and the integrated stress response[J].Molecular Cell,2010,40(2):280-293
[28]Alverca E,Andrade M,Dias E,et al.Morphological and ultrastructural effects of microcystin-LR from Microcystis aeruginosa extract on a kidney cell line[J].Toxicon,2009,54(3):283-294
[29]Chen Y,Zhou Y,Wang X,et al.Microcystin-LR induces autophagy and apoptosis in rat sertoli cells in vitro[J].Toxicon,2013,76:84-93
[30]Li Y.The effects of ER stress and autophagy on the apoptosis induced by microcystin-LR in Chinese hamster ovary cells[D].Thesis for master of Science,Zhengzhou University.2016[李洋.内质网应激-自噬在微囊藻毒素-LR诱导Cho细胞凋亡中的作用.硕士学位论文,郑州大学.2016]