Cloning and transcriptional regulation of slitrk3 gene promoter in large yellow croaker
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摘要: 神经突触相关蛋白Slitrk3具有调节抑制性突触发育的作用。研究大黄鱼 (Larimichthys crocea) 神经突触黏附分子slitrk3基因的转录调控机制，可为解决大黄鱼养殖面临的生长、应激及抗逆等问题提供新思路。通过生物信息学方法对大黄鱼及其他物种的Slitrk3氨基酸进行多重序列比对和系统进化树分析，并预测大黄鱼slitrk3基因启动子区域相关的调控元件，采用双荧光素酶报告基因系统检测大黄鱼slitrk3基因启动子区域的转录活性。生物信息学分析结果显示，Slitrk3氨基酸序列在鱼类中具有较高的保守性；大黄鱼slitrk3基因启动子区域预测存在2个潜在的转录起始位点、2个CpG岛以及Sp1、GR、C/EBPα和C/EBPβ等多种转录因子结合位点。双荧光素酶报告基因系统结果显示，大黄鱼slitrk3基因启动子区域的−1970—−1614 bp、−1210—−667 bp存在正调控元件，−1614—−1210 bp、−667—−376 bp、−376—−147 bp存在负调控元件，推测−147—+16 bp为核心启动子。Abstract: Slitrk3, a neurosynaptic-related protein, can regulate the development of inhibitory synapses. To explore the transcriptional regulation mechanism of slitrk3 gene of the large yellow croaker (Larimichthys crocea) can provide a new idea for solving the problems of growth, stress and anti-stress in the L. crocea culture. We conducted a multiple sequence alignment and a phylogenetic tree analysis of amino acids for Slitrk3 in L. crocea and other species to investigate the transcriptional regulation mechanism of the neural cell adhesion molecule for slitrk3 gene. Besides, we predicted the potential core promoter regions, CpG islands and transcription factor binding sites of slitrk3 gene by bioinformatics methods, and detected the Luciferase activity of promoter of slitrk3 gene by the Dual-Luciferase Reporter System. Bioinformatics analysis shows that the amino acid sequences of Slitrk3 were highly conserved in fish. There were two transcription start sites, two CpG islands, and multiple transcription factor binding sites such as Sp1, GR, C/EBPα and C/EBPβ in promoter of slitrk3 gene. Dual-Luciferase Reporter System shows that the regions from −1970 to −1614 bp and from −1 210 to −667 bp contained positive regulatory elements; while the regions from −1614 to −1210 bp, from −667 to −376 bp and from −376 to −147 contained negative regulatory elements; and the regions from −147 to +16 bp might be core promoter of slitrk3 gene. The results lay a theoretical foundation for the further study of transcriptional regulation mechanism of slitrk3 gene in L. crocea.
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Key words:
- Larimichthys crocea /
- slitrk3 gene /
- Promoter /
- Transcriptional regulation
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图 1 大黄鱼及其他物种的Slitrk3氨基酸序列多重序列比对
Figure 1. Multiple alignments of amino acid sequences of Slitrk3 in L. crocea and other species
图 2 大黄鱼及其他物种的Slitrk3氨基酸序列系统进化树
Figure 2. Phylogenetic tree of amino acid sequences of Slitrk3 in L. crocea and other species
图 3 大黄鱼slitrk3基因启动子CpG岛的预测
Figure 3. Prediction of CpG islands of slitrk3 gene promoter in L. crocea
图 4 大黄鱼slitrk3基因启动子不同长度片段的PCR产物电泳
注：M. DL2000 DNA Marker；1. slitrk3-P1片段；2. slitrk3-P2片段；3. slitrk3-P3片段；4. slitrk3-P4片段；5. slitrk3-P5片段；6. slitrk3-P6片段。
Figure 4. Electrophoresis of PCR products of slitrk3 gene promoter different length fragments in L. crocea
Note: M. DL2000 DNA Marker; 1. slitrk3-P1 fragment; 2. slitrk3-P2 fragment; 3. slitrk3-P3 fragment; 4. slitrk3-P4 fragment; 5. slitrk3-P5 fragment; 6. slitrk3-P6 fragment.
图 5 大黄鱼slitrk3基因启动子重组质粒酶切电泳
注：M. DL5000 DNA Marker；0. pGL3-Basic质粒；1. pGL3-slitrk3-P1质粒；2. pGL3-slitrk3-P2质粒；3. pGL3-slitrk3-P3质粒；4. pGL3-slitrk3-P4质粒；5. pGL3-slitrk3-P5质粒；6. pGL3-slitrk3-P6质粒。
Figure 5. Electrophoresis of restriction enzymes digestion of slitrk3 gene promoter recombinant plasmids in L. crocea
Note: M. DL5000 DNA Marker; 0. pGL3-Basic plasmid; 1. pGL3-slitrk3-P1 plasmid; 2. pGL3-slitrk3-P2 plasmid; 3. pGL3-slitrk3-P3 plasmid; 4. pGL3-slitrk3-P4 plasmid; 5. pGL3-slitrk3-P5 plasmid; 6. pGL3-slitrk3-P6 plasmid.
图 6 大黄鱼slitrk3基因启动子不同长度片段活性分析
注：数据均以平均值±标准误表示 (每个样品3次重复)；不同字母表示差异极显著 (P<0.01)；*. 差异显著 (P<0.05)。
Figure 6. Activity analysis of different length fragments of slitrk3 gene promoter in L. crocea
Note: Data are described as $ \overline { X}\pm { \rm {SD}}$1 (N=3); different letters above the bars indicate extremely significant differences at P<0.01; *. Significant differences at P<0.05.
表 1 大黄鱼及其他物种的Slitrk3氨基酸序列的GeneBank登录号
Table 1. GeneBank ID of amino acid sequences of Slitrk3 in L. crocea and other species
物种
SpeciesGeneBank登录号
GeneBank ID大黄鱼 Larimichthys crocea XP_010750353.1 棘头梅童鱼 Collichthys lucidus TKS81208.1 金钱鱼 Scatophagus argus XP_046245335.1 大西洋鲷 Sparus aurata XP_030298892.1 黃鲈 Perca flavescens XP_028430026.1 大鳍弹涂鱼 Periophthalmus magnuspinnatus XP_033832314.1 斑马鱼 Danio rerio XP_021323398.1 大鼠 Rattus norvegicus NP_001101153.1 小鼠 Mus musculus NP_001344780.1 智人 Homo sapiens NP_001305739.1 
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表 2 大黄鱼slitrk3基因启动子不同长度片段的扩增引物
Table 2. Primers used for fragments amplification primers of L. crocea slitrk3 gene
引物
Primer序列 (5'—3')
Sequence (5'−3')扩增区域
Amplification region/bp产物大小
Amplification size/bpslitrk3-P1 F: ctatcgataggtaccGAGCTCATCCGTAGCTCATTCACATGC −1970—+16 2029 R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG slitrk3-P2 F: ctatcgataggtaccGAGCTCGCGGAATCAATCATTTCTGGA −1614—+16 1673 R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG slitrk3-P3 F: ctatcgataggtaccGAGCTCGAATCCTCGATCAGCCGATGT −1210—+16 1269 R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG slitrk3-P4 F: ctatcgataggtaccGAGCTCCAAACTGACACCTATTTTTCC −667—+16 726 R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG slitrk3-P5 F: ctatcgataggtaccGAGCTCTGTCTGAGTGGAGAGATTTGT −376—+16 435 R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG slitrk3-P6 F: ctatcgataggtaccGAGCTCCTTTGTGAGGGTGTGTGTATC −147—+16 206 R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG 注：单下划线为Sac I酶切位点，下划线为Hind III酶切位点，小写字母为质粒同源序列。 Note: The single underline are the Sac I restriction enzyme digestion sites; the underline is the Hind III restriction enzyme digestion site; and the lowercase letters are the plasmid homologous sequences.
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表 3 大黄鱼slitrk3基因启动子转录因子结合位点的预测
Table 3. Prediction of transcription factor binding sites of slitrk3 gene promoter in L. crocea
转录因子
Transcription factor起始
Star/bp终止
End/bp序列 (5'—3')
Sequence (5'−3')Sp1 −1 901 −1 889 gtgcccccccctc −1 422 −1 413 gctcaggcca −1 037 −1 028 cgcccactgc −646 −637 ccgtcctctt −520 −511 gtctcctcac −504 −495 cctcacccac −145 −136 tgtgagggtg −107 −98 tccctccttt −84 −75 cccatcccct GR −1 893 −1 884 ccctctgttc −1 801 −1 792 aacagaacac −78 −69 ccctctgttc C/EBPα −1 697 −1 688 ttattttttt −551 −542 tgttatgtaa −361 −352 tttgtttgca −312 −303 tatattttgt −67 −58 gttttgcagt C/EBPβ −979 −970 attggaaaat GATA-1 −1 735 −1 726 gacatgataa TBP −1 691 −1 682 tttttagatt TEC1 −1 577 −1 568 cggaatgaaa RAP1 −1 327 −1 318 gtgtttgtgt NF-1 −757 −748 tggcacccat MEB-1 −698 −689 ttatttttaa GLO −698 −689 ttatttttaa E1 −498 −489 ccacctgctg Myf-3 −498 −489 ccacctgctg Oct-1 −339 −330 aaattatcca IRF-1 −72 −63 gttctgtttt
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[1] ARUGA J, MIKOSHIBA K. Identification and characterization of Slitrk, a novel neuronal transmembrane protein family controlling neurite outgrowth[J]. Mol Cell Neurosci, 2003, 24(1): 117-129. doi: 10.1016/S1044-7431(03)00129-5 [2] ARUGA J, YOKOTA N, MIKOSHIBA K. Human SLITRK family genes: genomic organization and expression profiling in normal brain and brain tumor tissue[J]. Gene, 2003, 315: 87-94. doi: 10.1016/S0378-1119(03)00715-7 [3] CHEN C Y, MILLION M, ADELSON D W, et al. Intracisternal urocortin inhibits vagally stimulated gastric motility in rats: role of CRF(2)[J]. Br J Pharmacol, 2002, 136(2): 237-247. doi: 10.1038/sj.bjp.0704713 [4] 靳雁斌, 范文红, 范明. Slitrk基因家族的研究进展[J]. 国际病理科学与临床杂志, 2006(2): 159-161. [5] GOMEZ-CASTRO F, ZAPPETTINI S, PRESSEY J C, et al. Convergence of adenosine and GABA signaling for synapse stabilization during development[J]. Science, 2021, 374(6568): k2055. doi: 10.1126/science.abk2055 [6] ROUND J, ROSS B, ANGEL M, et al. Slitrk gene duplication and expression in the developing zebrafish nervous system[J]. Dev Dyn, 2014, 243(2): 339-349. doi: 10.1002/dvdy.24076 [7] AKERMAN C J, CLINE H T. Refining the roles of GABAergic signaling during neural circuit formation[J]. Trends Neurosci, 2007, 30(8): 382-389. doi: 10.1016/j.tins.2007.06.002 [8] 刘鸷驹, 张东星, 晏仁义, 等. γ-氨基丁酸的生物活性研究进展[J]. 现代药物与临床, 2022, 37(9): 2167-2172. [9] 彭思博, 陈秀梅, 孔雨昕, 等. γ-氨基丁酸对乌鳢生长性能、肠道消化酶和抗氧化酶活性的影响[J]. 饲料工业, 2020, 41(14): 40-45. doi: 10.13302/j.cnki.fi.2020.14.009 [10] 伍广涛. γ-氨基丁酸及胍基乙酸对锦鲤生长性能及抗运输应激的影响[D]. 大连: 大连海洋大学, 2022: 13-17. [11] 彭思博, 陈秀梅, 王桂芹. γ-氨基丁酸的代谢及其在鱼类养殖中的应用研究进展[J]. 中国畜牧杂志, 2020, 56(7): 22-27. doi: 10.19556/j.0258-7033.20190816-07 [12] DANINO Y M, EVEN D, IDESES D, et al. The core promoter: at the heart of gene expression[J]. Biochim Biophys Acta, 2015, 1849(8): 1116-1131. doi: 10.1016/j.bbagrm.2015.04.003 [13] SLOUTSKIN A, SHIR-SHAPIRA H, FREIMAN R N, et al. The core promoter is a regulatory hub for developmental gene expression[J]. Front Cell Dev Biol, 2021, 9: 666508. doi: 10.3389/fcell.2021.666508 [14] 刘相莲, 乔芳, 黄德玉, 等. 转录因子研究方法进展[J]. 生理科学进展, 2017, 48(1): 73-76. doi: 10.3969/j.issn.0559-7765.2017.01.019 [15] KAJIWARA Y, BUXBAUM J D, GRICE D E. SLITRK1 binds 14-3-3 and regulates neurite outgrowth in a phosphorylation-dependent manner[J]. Biol Psychiatry, 2009, 66(10): 918-925. doi: 10.1016/j.biopsych.2009.05.033 [16] KATAYAMA K, ZINE A, OTA M, et al. Disorganized innervation and neuronal loss in the inner ear of Slitrk6-deficient mice[J]. PLoS One, 2009, 4(11): e7786. doi: 10.1371/journal.pone.0007786 [17] ABELSON J F, KWAN K Y, O'ROAK B J, et al. Sequence variants in SLITRK1 are associated with Tourette's syndrome[J]. Science, 2005, 310(5746): 317-320. doi: 10.1126/science.1116502 [18] SHMELKOV S V, HORMIGO A, JING D, et al. Slitrk5 deficiency impairs corticostriatal circuitry and leads to obsessive-compulsive-like behaviors in mice[J]. Nat Med, 2010, 16(5): 598-602. doi: 10.1038/nm.2125 [19] PROENCA C C, GAO K P, SHMELKOV S V, et al. Slitrks as emerging candidate genes involved in neuropsychiatric disorders[J]. Trends Neurosci, 2011, 34(3): 143-153. doi: 10.1016/j.tins.2011.01.001 [20] 王媛, 肖泽萍. Slitrk家族在精神疾病中作用的研究现状[J]. 上海交通大学学报 (医学版), 2014, 34(9): 1402-1405. [21] TAKAHASHI H, KATAYAMA K, SOHYA K, et al. Selective control of inhibitory synapse development by Slitrk3-PTPdelta trans-synaptic interaction[J]. Nat Neurosci, 2012, 15(3): 389-398, S1-S2. doi: 10.1038/nn.3040 [22] FRITSCHY J M, TYAGARAJAN S K. GABAergic synaptogenesis: a case for cooperation[J]. Neuron, 2017, 96(4): 709-711. doi: 10.1016/j.neuron.2017.11.003 [23] LI J, HAN W, PELKEY K A, et al. Molecular dissection of neuroligin 2 and Slitrk3 reveals an essential framework for GABAergic synapse development[J]. Neuron, 2017, 96(4): 808-826. doi: 10.1016/j.neuron.2017.10.003 [24] LI J, HAN W Y, WU K W, et al. A conserved tyrosine residue in Slitrk3 carboxyl-terminus is critical for GABAergic synapse development[J]. Front Mol Neurosci, 2019, 12: 213. doi: 10.3389/fnmol.2019.00213 [25] 史玉杰, 李庆贺, 刘晓辉. DNA甲基化与基因表达调控研究进展[J]. 中国生物工程杂志, 2013, 33(7): 90-96. doi: 10.13523/j.cb.20130714 [26] 罗舒月. Sp蛋白家族对人IL10RB基因的转录调控作用[D]. 重庆: 重庆医科大学, 2021: 81-82. [27] LIU B, ZHANG T N, KNIGHT J K, et al. The glucocorticoid receptor in cardiovascular health and disease[J]. Cells, 2019, 8(10): 1227. DOI: 10.3390/cells8101227. [28] ZHANG X, CHE L, SHAN J, et al. The effects of Formoterol in preventing adipogenesis and obesity are mediated by PPARgamma/C/EBPalpha axis and AMPK/PGC-1alpha pathway[J]. Biosci Biotechnol Biochem, 2022: zbac103. DOI: 10.1093/bbb/zbac103. [29] WESOLOWSKI R, KOWENZ-LEUTZ E, ZIMMERMANN K, et al. Myeloid transformation by MLL-ENL depends strictly on C/EBP[J]. Life Sci Alliance, 2021, 4(1): e202000709. doi: 10.26508/lsa.202000709 [30] ZENG Y, LI F D, DU J L, et al. Potent antitumor activity of oncolytic adenovirus expressing C/EBPbeta against hepatocellular carcinoma[J]. Apoptosis, 2020, 25(3/4): 154-156. [31] CALELLA A M, NERLOV C, LOPEZ R G, et al. Neurotrophin/Trk receptor signaling mediates C/EBPalpha, -beta and NeuroD recruitment to immediate-early gene promoters in neuronal cells and requires C/EBPs to induce immediate-early gene transcription[J]. Neural Dev, 2007, 2: 4. doi: 10.1186/1749-8104-2-4 -
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