半滑舌鳎突触体相关蛋白29基因(<i>SNAP29</i>)克隆及其组织分布和时序表达分析

doi:10.7668/hbnxb.20194575

摘要/Abstract

摘要：

为了分析半滑舌鳎突触体相关蛋白29基因(SNAP29)的分子生物学特征、组织分布特点以及感染海藻希瓦氏菌后的时序表达规律,通过RT-PCR、生物信息学网站、qRT-PCR技术对半滑舌鳎SNAP29基因进行了扩增、生信分析和表达特征研究。结果表明,半滑舌鳎SNAP29基因CDS区长度为789 bp,编码262个氨基酸,理论等电点(pI)为5.39,分子质量为29.717 81 ku,有2个典型的SNARE结构域;二级结构主要由α-螺旋和无规卷曲组成,二级结构和三级结构基本一致;多序列比对结果显示,半滑舌鳎与欧洲鲈鱼SNAP29基因相似性最高;SNAP29基因在健康半滑舌鳎所测的各组织中均有表达,在鳃组织中表达最高,在脑组织中表达量最低;利用海藻希瓦氏菌人工感染半滑舌鳎后,SNAP29基因在肠道组织、心脏组织和脑组织中表达量主要呈现上调趋势,在肝脏、头肾、脾脏组织中主要呈现下调表达,SNAP29基因在健康半滑舌鳎组织中均有表达;海藻希瓦氏菌感染半滑舌鳎的肠道、脑、心脏、肝脏、头肾、脾脏组织中SNAP29基因呈现差异表达。综上,SNAP29可能参与机体免疫应答过程或病理生理过程。

关键词: 半滑舌鳎, SNAP29, 基因克隆, qRT-PCR, 海藻希瓦氏菌

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

吴茵茵, 温思怡, 韩卓然, 孙敬锋. 半滑舌鳎突触体相关蛋白29基因(SNAP29)克隆及其组织分布和时序表达分析[J]. 华北农学报, 2024, 39(2): 231-238. doi: 10.7668/hbnxb.20194575.

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.

http://www.hbnxb.net/CN/Y2024/V39/I2/231

图/表 10

表1 引物序列

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

图1 半滑舌鳎SNAP29基因PCR扩增产物琼脂糖凝胶电泳 M.DL2000 Marker;1.SNAP29基因PCR扩增产物。

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

图2 半滑舌鳎SNAP29编码氨基酸序列及蛋白结构域预测 A.SNAP29 基因的编码氨基酸序列,红色为起始密码子与终止密码子,下划线为结构域;B.SNAP29蛋白结构域。

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.

图3 半滑舌鳎SNAP29编码蛋白的疏/亲水性分析结果

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

图4 半滑舌鳎SNAP29氨基酸序列糖基化预测和磷酸化位点分析横坐标为氨基酸序列,纵坐标为阈值。

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.

图5 SNAP29蛋白的二级结构预测(A)与三级结构预测(B)

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

图6 半滑舌鳎SNAP29基因编码氨基酸序列同源性比对阴影区域.同源性氨基酸;黑色.同源性为100%;粉红色.同源性大于75%;浅蓝色.同源性大于50%;黄色.同源性大于33%。

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.

图7 SNAP29蛋白系统进化树

Fig.7 SNAP29 protein phylogenetic tree

图8 半滑舌鳎不同组织中SNAP29表达水平两组间分别比较,不同字母表明两组间差异显著(P<0.05)。

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).

图9 海藻希瓦氏菌感染后半滑舌鳎不同组织中SNAP29的表达变化不同字母表示不同时间差异达显著水平(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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半滑舌鳎突触体相关蛋白29基因(SNAP29)克隆及其组织分布和时序表达分析

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

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半滑舌鳎突触体相关蛋白29基因(SNAP29)克隆及其组织分布和时序表达分析

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

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