Cloning and Expression Analysis of TRAF6 from Large Yellow Croaker Larimichthys crocea

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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）

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

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

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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