大黄鱼TRAF6的克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1389

摘要/Abstract

摘要：

肿瘤坏死因子受体相关因子6（tumor necrosis factor receptor associated factor 6,TRAF6）是TRAF家族的重要成员,参与调控模式识别受体介导的信号通路,在宿主的固有免疫应答中发挥重要作用。本研究以大黄鱼（Larimichthys crocea）为实验对象,对大黄鱼TRAF6基因进行了克隆与鉴定。克隆获得的大黄鱼TRAF6基因ORF全长1 725 bp,编码574 aa,其基因结构由7个外显子和6个内含子构成。亚细胞定位分析结果表明大黄鱼TRAF6定位于细胞的胞浆,并在细胞核周围存在点状聚集现象。表达分析结果显示TRAF6广泛表达于健康大黄鱼不同组织/器官中,在血液中表达水平最高,在脑中表达水平最低;且在Poly I：C、LPS、PGN和变形假单胞菌（Pseudomonas plecoglossicida）刺激下,大黄鱼鳃、脾、头肾、肠与血液组织中TRAF6的表达水平显著上调。上述研究结果揭示了大黄鱼TRAF6的序列与时空表达特征,为深入解析大黄鱼TRAF6在宿主免疫反应中的作用及其分子机制提供了参考。

关键词: 大黄鱼, 肿瘤坏死因子受体相关因子6, 固有免疫, 表达分析

Abstract:

Tumor necrosis factor receptor-associated factor 6（tRAF6）is an important member of the TRAF family,which participate in the regulation of pattern recognition receptors mediated signaling pathways and play important roles in host innate immune response. In the present study,one ortholog of TRAF6 gene was cloned and identified in large yellow croaker（Larimichthys crocea）. The identified ORF of TRAF6 gene was 1 725 bp,encoding a protein of 574 aa,with the gene structure composed of 7 exons and 6 introns. Subcellular localization analysis showed that TRAF6 was located in the cytoplasm,and obvious aggregation was identified around the nucleus. Expression analysis indicated that TRAF6 was widely expressed in different tissues/organs of healthy large yellow croakers,with the highest expression in the blood and the lowest expression in the brain. Additionally,the mRNA expressions of TRAF6 in the gill,spleen,head kidney,intestine,and blood were significantly upregulated in response to Poly I：C,LPS,PGN and Pseudomonas plecoglossicida stimulations. In conclusion,the present study revealed the sequence as well as expression patterns of the TRAF6 in large yellow croaker,providing a reference for further investigation on the function and molecular mechanism that TRAF6 involved in host immune response.

Key words: Larimichthys crocea, TRAF6, innate immunity, expression analysis

陈英, 王艺磊, 邹鹏飞. 大黄鱼TRAF6的克隆及表达分析[J]. 生物技术通报, 2022, 38(8): 233-243.

CHEN Ying, WANG Yi-lei, ZOU Peng-fei. Cloning and Expression Analysis of TRAF6 from Large Yellow Croaker Larimichthys crocea[J]. Biotechnology Bulletin, 2022, 38(8): 233-243.

图/表 7

表1 本研究所用引物

Table 1 Primers used in this study

引物名称Primer name	引物序列Primer sequence（5'-3'）	目的Application
Lc-TRAF6-F	ATGGCTTGCATTGACAGCAAT	Lc-TRAF6 ORF cloning
Lc-TRAF6-R	AGCCTCGGTTTCAAGGGAC	Lc-TRAF6 ORF cloning
pTurbo-TRAF6-F	CCGGAATTCTGATGGCTTGCATTGACAGCAAT	pTurbo-TRAF6-GFP
pTurbo-TRAF6-R	CGCGGATCCCGGTCCCTTGAAACCGAGGCT	pTurbo-TRAF6-GFP
qTRAF6-F	GACGGACGGTTGGTAAAGCAG	RT-qPCR
qTRAF6-R	CAACTTGTAGCCTGGACGACCC	RT-qPCR
qβ-actin-F	TTATGAAGGCTATGCCCTGCC	RT-qPCR
qβ-actin-R	TGAAGGAGTAGCCACGCTCTGT	RT-qPCR

图1 大黄鱼TRAF6与其他脊椎动物TRAF6氨基酸序列比对黑色箭头分别代表环指结构域、锌指结构域、卷曲结构域和MATH结构域

Fig. 1 Multiple alignment of Lc-TRAF6 with TRAF6 in other vertebrates The black arrows represent the RING finger domain,zinc finger domain, coiled-coil domain, and MATH domain respectively

表2 Lc-TRAF6与其它脊椎动物TRAF6氨基酸序列相似性比较

Table 2 Comparison of amino acid sequence similarity between Lc-TRAF6 and TRAF6 in other vertebrates

常用名Common name	物种名Scientific name	NCBI序列登录号Accession No.	序列长度Length/aa	一致性Identity/%	相似性Similarity/%
Grouper	Epinephelus coioides	AGQ45557.1	570	89	94
Fugu	Takifugu rubripes	XP_011608207.2	564	79	87
Rainbow trout	Oncorhynchus mykiss	AVM80404.1	551	66	78
Grass carp	Ctenopharyngodon idella	AGI51678.1	542	63	75
Zebrafish	Danio rerio	NP_001038217.1	542	61	74
channel catfish	Ictalurus punctatus	XP_017313923.1	540	60	74
Chicken	Gallus gallus	XP_015142694.1	545	52	66
Human	Homo sapiens	NP_665802.1	522	51	65
Mouse	Mus musculus	NP_001290202.1	530	50	64

图2 Lc-TRAF6与其他脊椎动物TRAF6基因结构比较大黄鱼、红鳍东方鲀、斑马鱼、鸡、小鼠和人TRAF6基因的外显子和内含子结构比较。外显子和内含子分别用黑色方框和线条表示,外显子的长度大小显示在黑框的上方,而内含子的长度大小则显示在线条下方。黑框的长度以及线的长度与其序列长度成正比。基因序列信息及其GenBank数据库登录号：大黄鱼,NC_040018.1（4990344-4997110）;红鳍东方鲀,NC_042297.1（13166964-13171483）;斑马鱼,NC_007118.7（48722838-48738718）;鸡,NC_052536.1（19015766-19036372）;小鼠,NC_000068.8（101508765-101532013）;人,NC_000011.10（36483769-36510313）

Fig.2 Genomic structure comparison of gene Lc-TRAF6 with TRAF6 in other vertebrates Structure comparison of exons and introns of TRAF6 gene in L. crocea,T. rubripes,D. rerio,G. gallus,M. musculus and H. sapiens. Exons and introns are represented by black boxes and lines respectively,with the length of the exon shown above the black box and the length of the intron shown below the line. The length of the black box and the length of the line is proportional to the length of the sequence. Gene sequences information and their GenBank accession numbers are shown as follows：L. crocea,NC_040018.1（4990344-4997110）;T. rubripes,NC_042297.1（13166964-13171483）;D. rerio,NC_0071187（48722838-48738718）;G. gallus,NC_052536.1（19015766-19036372）;M. musculus,NC_000068.8（101508655-101532013）;H. sapiens,NC_000011.10（36483769-36510313）

图3 Lc-TRAF6的亚细胞定位分析分别用pTurboGFP和pTurbo-TRAF6-GFP瞬时转染HEK 293T细胞。在转染24 h后,用DAPI进行细胞核染色,在激光共聚焦显微镜下观察细胞并拍照

Fig.3 Subcellular localization analysis of Lc-TRAF6 HEK 293T cells were transiently transfected with pTurboGFP and pTurbo-TRAF6-GFP respectively. At 24 h after transfection,cells were stained with DAPI,detected under a confocal microscope and photographed

图4 Lc-TRAF6的组织表达分析实时荧光定量PCR检测了健康大黄鱼在11种组织/器官中Lc-TRAF6 mRNA表达情况。将Lc-TRAF6 mRNA在脑中的表达水平设定为1倍,在其他器官/组织中Lc-TRAF6 mRNA的表达水平记录为相对于脑中表达水平的倍数,1倍的基线用红色虚线标记。所有数据均以平均值±标准误表示（n = 6）。组间差异无统计学意义用相同上标,统计学上差异显著用不同的上标表示（P < 0.05）

Fig. 4 Tissue expression analysis of Lc-TRAF6 The expression pattern of Lc-TRAF6 mRNA in 11 different tissues/organs of healthy large yellow croaker was detected by quantitative real-time PCR analysis. The mRNA expression level of Lc-TRAF6 in the brain was set as 1-fold,and the mRNA expression level of Lc-TRAF6 in other organs/tissues was recorded as multiple of the expression level in the brain,and the baseline of 1-fold was marked with red dotted line. All data were expressed as mean±SE（n = 6）. Different superscripts indicate statistically different results（P < 0.05）and the same superscript indicates no statistical differences between groups

图5 Poly I：C、LPS、PGN和P. plecoglossicida刺激下Lc-TRAF6的表达分析 Poly I：C、LPS、PGN和P. plecoglossicida免疫刺激健康大黄鱼6、12、24 h后（以PBS为对照）,通过实时荧光定量PCR技术检测鱼鳃（A）、脾脏（B）、头肾（C）、肠（D）和血液（E）中Lc-TRAF6 mRNA的表达水平。其结果以β-actin为内参基因进行校正,数据表示为平均值±标准误（n=6）,* P <0.05,** P <0.01

Fig. 5 Expression analysis of Lc-TRAF6 under Poly I：C,LPS,PGN,and P. plecoglossicida stimulation The healthy large yellow croakers was stimulated with Poly I：C,LPS,PGN,and P. plecoglossicida for 6,12,and 24 h（with PBS set as the control group）,then the mRNA expression levels of Lc-TRAF6 in gill（A）,spleen（B）,head kidney（C）,intestine（D）,and blood（E）were detected by RT-qPCR. The results were normalized to the expression of β-actin and the data were recorded as mean ±SE（n = 6）. * P < 0.05,**P < 0.01

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