MOLECULAR CLONING AND FUNCTION ANALYSIS OF TACHYSURUS FULVIDRACO FTR67
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摘要: 鱼类TRIM(Tripartite motif)家族的特异性扩张产生了一个硬骨鱼类特有的亚家族finTRIM (Fish novel TRIM, FTR), 为探究finTRIM在黄颡鱼(Tachysurus fulvidraco)先天抗病毒免疫中发挥的作用, 文章鉴定了1个与斑马鱼(Danio rerio) FTR67同源性较高的黄颡鱼finTRIM基因, 命名为TfFTR67 (Tachysurus fulvidraco FTR67)。系统进化树分析表明, FTR67在有些鱼类物种中发生了基因重复, 导致基因拷贝增多。TfFTR67不受SVCV的诱导表达, 是一个组成型表达的基因。过量表达TfFTR67抑制poly(I﹕C)及RLR信号分子诱导的干扰素反应, 同时促进病毒在细胞中的复制。鉴于TfFTR67与斑马鱼FTR67的表达特征和功能不同, 研究结果表明, 鱼类FTR67在不同物种中发生了基因重复和功能歧化。Abstract: TRIM (Tripartite motif) proteins play direct antiviral and indirect regulatory roles in vertebrate innate antiviral responses. The independent expansion of fish TRIM family gives rise to a subfamily unique to teleosts, named finTRIM (fish novel TRIM, FTR). In the current study, we identified a yellow catfish finTRIM gene, which is most homologous to zebrafish (Danio rerio) FTR67, thus named TfFTR67 (Tachysurus fulvidraco FTR67). Phylogenetic tree analysis showed that FTR67 has undergone gene duplication in some fish species, resulting in increased gene copies. RT-PCR showed that TfFTR67 is virally inducible and thus is a constitutively expressed gene. Overexpression of TfFTR67 inhibited the interferon response induced by poly(I﹕C) transfection and overexpression of RLR signaling molecules, and also promoted virus replication in fish cells. A previous report has shown that zebrafishFTR67 is induced by virus infection, and overexpression of zebrafish FTR67 enhances fish interferon response. Therefore, yellow catfish FTR67 displays the expression characteristics and function features that are different from those of zebrafish FTR67. In addition, three copies of FTR67 are found in goldfish genome, indicating that there is independent expansion of FTR67 gene in certain fish species. Actually, we do not know whether there is only one copy of FTR67 gene in yellow catfish genome although a single one is found up to now. These data together suggest that fish FTR67 has undergone gene duplication and functional diversification after radiation of fish species.
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Keywords:
- finTRIM /
- Independent expansion /
- Interferon response /
- Negative regulation /
- Tachysurus fulvidraco
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图 1 FTR67的氨基酸序列及蛋白结构域组成分析
A. 黄颡鱼、斑马鱼、苏氏圆腹𩷶、草鱼FTR67与鲫FTRCA1和斑马鱼FTR42蛋白的多重序列比对; 结构域用黑框表示; (*)和(.和:)分别表示氨基酸的一致性和相似性; B. 黄颡鱼、斑马鱼、苏氏圆腹𩷶、草鱼FTR67与鲫FTRCA1、斑马鱼FTR42的蛋白结构域组成
Figure 1. Multiple alignments and domain arrangements of FTR67s
A. Multiple alignments of several fish FTR67 proteins and crucian carp-specific FTRCA1 and zebrafish FTR42. The domains of protein are indicated by boxes. The identical (*) and similar (. and :) amino acids are indicated; B. Schematic representation of FTR67 from yellow catfish, striped catfish, zebrafish and goldfish, crucian carp FTRCA1 and zebrafish FTR42 proteins
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图 2 黄颡鱼FTR67的系统进化树分析
利用MEGA X软件进行多重序列比对, 在此基础上运用邻近法构建系统进化树
Figure 2. The evolutionary relationship of yellow catfish FTR67 with other finTRIM proteins
A neighbor-joining tree is constructed based on analysis of protein sequences using MEGA X program
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图 3 黄颡鱼、苏氏圆腹?、斑马鱼和金鱼FTR67基因座位的共线性分析
通过BLAST分析并结合进化树分析, 三个粗体TRIM25L为金鱼FTR67同源基因, 黑框中的TRIM16L和TRIM25L实际上是FTR87的同源基因, GMRF35L是PIGRL的同源基因
Figure 3. Syntenic analyses of FTR67 gene loci from yellow catfish, striped catfish, zebrafish and goldfish
BLAST searches combined with phylogenetic tree analyses show that three goldfish TRIM25Ls in bolds are the orthologs of FTR67, the TRIM25L and TRIM16L in boxes are the orthologs of FTR87, and GMRF35L is the ortholog of PIGRL
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图 4 TfFTR67在黄颡鱼组织中的表达
A. RT-PCR分析TfFTR67、IFN和Mx1基因在SVCV病毒攻毒前后的黄颡鱼12种组织中的转录表达; B. TfFTR67基因的5′侧翼序列; 粗体表示NF-κB结合位点, 斜体表示SP1结合位点, 黑框表示起始密码子, 下划线表示TfFTR67基因的5′非翻译区; C. TfFTR67基因启动子中转录因子结合位点组成; 数字表示相对于TfFTR67基因的转录起始位点的碱基对位置
Figure 4. Expression analysis of TfFTR67 in yellow catfish tissues injected with or without SVCV
A. RT-qPCR analysis of TfFTR67, IFN and Mx1 transcription in 12 tissues of yellow catfish infected with and without SVCV; B. 5′ flanked sequence of TfFTR67 gene showing the predicated sites specific to different transcription factors, including NF-κB and SP1. The transcription start site is indicated by box; C. Schematic of TfFTR67 promoters showing the location of transcription factor binding sites. Numbers indicate the positions of base pairs relative to the transcription start site of TfFTR67
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图 5 过量表达TfFTR67抑制干扰素反应
A. 黄颡鱼IFN基因的5′侧翼序列; 黑框表示预测的ISRE基序, 下划线序列用于克隆TfIFN启动子序列构建TfIFNpro-luc质粒的引物, 黑框粗体字表起始密码子; B. TfIFNpro-luc表达质粒示意图; 数字表示相对于转录起始位点碱基对的位置; C. 转染poly(I﹕C)及过量表达RLR信号分子激活黄颡鱼IFN启动子的活性; D和E. 过量表达TfFTR67抑制胞内poly(I﹕C)诱导的TfIFNpro-luc的激活(D)及DrIFNφ1pro-luc的激活(E); F. 过量表达TfFTR67抑制RLR信号分子对DrIFNφ1pro-luc的激活
Figure 5. Overexpression of TfFTR67 negatively regulates IFN response
A. 5′ flanked sequences of yellow catfish IFN gene. The putative ISRE motifs are indicated in box, and the underlined highlights the primer for cloning TfIFN promoter DNA to construct TfIFN-pro-luc. The transcription start site is indicated by box and in bold; B. Schematic of TfIFNpro-luc. Numbers indicate the positions of base pairs relative to the transcription start site of TfIFN; C. Yellow catfish IFN promoter is activated by poly (I﹕C) transfection and overexpression of each of RLR signaling molecules; D and E. Overexpression of TfFTR67 inhibit the poly (I﹕C)-triggered activation of TfFTR67pro-luc (D) and DrIFNφ1pro-luc (E); F. Overexpression of TfFTR67 inhibit the activation of DrIFNφ1pro-luc by RLR signaling molecules
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图 6 过量表达TfFTR67促进病毒复制
在EPC细胞中过量表达TfFTR67, 12h后感染SVCV(5×103 TCID50/mL)。继续培养72h后, 通过结晶紫染色观察细胞CPE(A), 检测细胞上清液中的病毒滴度(B), RT-PCR检测细胞中IFN和Mx1基因的转录(C)
Figure 6. Overexpression of TfFRT67 promotes viral replication
EPC cells transfected with TfFTR67 are infected with SVCV (5×103TCID50/mL), followed by crystal violet staining observation (A), viral title detection of supernatants (B), and RT-PC detection of IFN and Mx1 transcripts (C)
表 1 实验所用引物
Table 1 Primers used in this experiment
引物名称
Primer序列
Sequence (5′—3′)TfFTR67-F CAGCCTGTCACATCGAGAC TfFTR67-R CCAAAGCTGAGAGATGCCTTAG TfFTR67-F-hm GCCACTGTGCTGGATgccaccATGGCGCAGGCGGGG TfFTR67-R-hm TGGAATTCTGCAGATCTACCGCCGTCTTCTCCG TfIFNpro-F-hm GAGCTCTTACGCGTGgccaccGTCGGGTGATGTTCACA TfIFNpro-R-hm CTCGAGCCCGGGCTAGCATGTTCTCGCTCTCTGCTCG TfFTR67-RT-F CCTTCCTGCAGAGCTACCGG TfFTR67-RT-R GTATTTGACTCCGCATCAGCC TfIFN-RT-F GATCGATAAGGCCAACACAG TfIFN-RT-R CAGTGTCCTGCTGTCCCA TfMx1-RT-F GCGCGAGTCTAAGTGAACAG TfMx1-RT-R AGCTCGAGTGGACATCTTGT Tfactin-RT-F GTCCGTGACATCAAGGAGAAGC Tfactin-RT-R AGGAGGAAGAGGCAGCAGTG EPCIFN-RT-F ATGAAAACTCAAATGTGGACGTA EPCIFN-RT-R GATAGTTTCCACCCATTTCCT EPCMx1-RT-F GGCTGGAGCAGGTGTTGGTATC EPCMx1-RT-R TCCACCAGGTCCGGCTTTGT EPCactin-RT-F CAGATCATGTTTGAGACC EPCactin-RT-R ATTGCCAATGGTGATGAC 
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