Cloning,Tissue Distribution and Temporal Expression Analysis of Synaptosome-associated Protein 29 Gene(SNAP29) in Cynoglossus semilaevis

doi:10.7668/hbnxb.20194575

Abstract

Abstract:

The aim of this study was to analyze synaptosome associated protein 29 gene(SNAP29)in Cynoglossus semilaevis.The molecular biological characteristics,tissue distribution characteristics of SNAP29 gene and the temporal expression pattern after infection with Shewanella algae.The SNAP29 gene of C.semilaevis was amplified by RT-PCR,bioinformatics website and qRT-PCR,and its bioinformatics analysis and expression characteristics were studied.The results showed that the CDS region of SNAP29 gene was 789 bp in length,encoding 262 amino acids.The theoretical isoelectric point(pI)was 5.39,and the molecular weight was 29.717 81 ku.There were two typical SNARE domains.The secondary structure was mainly composed of α-helix and random coil,and the secondary structure and tertiary structure were basically the same.The results of multiple sequence alignment showed that the SNAP29 gene of C.semilaevis had the highest similarity with that of European perch.The SNAP29 gene was expressed in all tissues of healthy C.semilaevis,with the highest expression in gill tissue and the lowest expression in brain tissue.After artificial infection of C.semilaevis with S.algae,the expression of SNAP29 gene was mainly up-regulated in intestinal tissue,heart tissue and brain tissue,and down-regulated in liver,head kidney and spleen tissue.SNAP29 gene was expressed in heart,brain,liver,intestinal tract,spleen,gill,middle kidney and head kidney of healthy C.semilaevis.The SNAP29 gene was differentially expressed in the intestine,brain,heart,liver,head kidney and spleen of C.semilaevis infected with S.algae.The above results indicate that SNAP29 may be involved in the body's immune response process or pathophysiological process.

Key words: Cynoglossus semilaevis, SNAP29, Genetic cloning, qRT-PCR, Shewanella algae

WU Yinyin, WEN Siyi, HAN Zhuoran, SUN Jingfeng. Cloning,Tissue Distribution and Temporal Expression Analysis of Synaptosome-associated Protein 29 Gene(SNAP29) in Cynoglossus semilaevis[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 231-238. doi: 10.7668/hbnxb.20194575.

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Figures/Tables 10

Tab.1 Primer sequence

引物名称 Primer name	引物序列(5'—3') Primer sequence (5'—3')	退火温度/℃ Annealing temperature	扩增长度/bp Expected size	用途 Purpose
SNAP29-F	ATAACGTCGTCTCTCGCTGC	55	900	PCR
SNAP29-R	GCGGACCGATCTGTTTGTCT
SNAP29Q-F	GAAGAGAACTGACAAGATGATG	50	81	qPCR
SNAP29Q-R	ACACTCTTGATGCTGCTAAT
β-actin-F	GTAGGTGATGAAGCCCAGAGCA	58	204	qPCR
β-actin-R	CTGGGTCATCTTCTCCCTGT

Fig.1 Agarose gel electrophoresis of PCR amplification products of SNAP29 gene in Cynoglossus semilaevis M.DL2000 Marker;1.SNAP29 gene PCR product.

Fig.2 Prediction of amino acid sequence and protein domain of SNAP29 in Cynoglossus semilaevis A.Amino acid sequence encoded by SNAP29 gene,red is the start codon and the stop codon,and the underline is the domain;B.SNAP29 protein domain.

Fig.3 Hydrophobicity/hydrophilicity analysis of SNAP29 protein in Cynoglossus semilaevis

Fig.4 Glycosylation prediction and phosphorylation site analysis of SNAP29 amino acid sequence in Cynoglossus semilaevis The abscissa is the amino acid sequence, and the ordinate is the threshold.

Fig.5 Secondary structure prediction(A)and tertiary structure prediction(B)of SNAP29 protein

Fig.6 Amino acid sequence homology comparison of the SNAP29 gene of Cynoglossus semilaevis Shaded areas.Homologous amino acids;Black color.100% homology;Pink.>75% homology; Light blue.>50% homology;Yellow.>33% homology.

Fig.7 SNAP29 protein phylogenetic tree

Fig.8 The expressions of SNAP29 in different tissues of Cynoglossus semilaevis Comparisons between the two groups were made separately,with different letters indicating significant differences between the two groups(P<0.05).

Fig.9 Temporal expression patterns of SNAP29 in different tissues of Cynoglossus semilaevis after infection with S.algae Different letters indicate differences at different time at a significant level(P<0.05).

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