杉虎斑Galectin-8基因克隆及其在不同阿魏酸水平饲料下的表达响应

doi:10.13560/j.cnki.biotech.bull.1985.2022-0059

摘要/Abstract

摘要：

Galectin-8是半乳糖凝集素家族的成员，对β-半乳糖苷类能够特异性识别，具有多种生物学功能。为研究杉虎斑（Epinephelus fuscoguttatus ♀× E. polyphekadion ♂）Galectin-8基因的序列特征、组织表达情况与饲料中添加阿魏酸（ferulic acid，FA）对其表达的影响，使用cDNA末端快速扩增技术（rapid amplification of cDNA ends，RACE）首次克隆得到了其全长cDNA序列。该序列长1 368 bp，其中5'UTR为16 bp，3'UTR为394 bp，开放阅读框（ORF）长960 bp，共编码319个氨基酸。该氨基酸序列在N端和C端上各具有一个糖识别结构域（carbohydrate binding domain，CRD），分别由136和133个氨基酸组成，由长为36个氨基酸的连接肽所连接，并包含两个保守基序H-NPR和WG-EE。同源性与系统发育进化树分析表明，杉虎斑Galectin-8与鞍带石斑鱼的一致性最高，为98.43%，并与其他硬骨鱼类聚为一支。RT-qPCR结果显示，Galectin-8在杉虎斑脾脏中表达量最高，其次是中肾、心脏、肠道、肝脏、头肾和鳃，而在脑、胃、皮肤和肌肉组织中微量表达。饲料中添加40-320 mg/kg 的FA时，肠道、肝脏、头肾和脾脏的Galectin-8表达量显著上调（P < 0.05），这表明FA可作为免疫刺激剂增强杉虎斑的先天性免疫。然而，杉虎斑Galectin-8的免疫功能尚不清，需要进一步的研究来阐明，以了解其在抵抗病原体入侵中的作用。

关键词: 杉虎斑, Galectin-8, 阿魏酸, 基因克隆, 表达分析

Abstract:

Galectin-8 is a member of the galectin family，and binds with β-galactoside by specific recognition with many biological functions. The present study was conducted to investigate Galectin-8 sequence characterization，its tissue distribution，and the effect of dietary ferulic acid（FA）supplementation on the Galectin-8 gene expression in hybrid grouper（Epinephelus fuscoguttatus ♀ × E. polyphekadion ♂）. The technology of rapid amplification of cDNA ends（RACE）was utilized to obtain the full length（1 368 bp）of hybrid grouper Galec-tin-8 cDNA for the first time. The cDNA was composed of 5' untranslated region（5' UTR）（16 bp），3' UTR（394 bp），and open reading frame（ORF）of 960 bp that encoded 319 amino acids. The amino acid sequence of galectin-8 possessed two carbohydrate recognition domains（CRDs）at N- and C- terminus composed of 136 and 133 amino acids，respectively，which were connected by a linker peptide of 36 amino acids and contained two conserved H-NPR and WG-EE motifs. Homology and phylogenetic tree analysis indicated that hybrid grouper Galectin-8 demonstrated the highest identity（98.43%）with E. fuscoguttatus，and was clustered with other teleosts. Based on RT-qPCR，tissue distribution analysis revealed that Galectin-8 showed the highest expressions in the spleen，followed by mid kidney，heart，intestine，liver，head kidney，and gill，yet the trace expression in the brain，stomach，skin，and muscle. Moreover，dietary FA supplementation at level of 40-320 mg/kg considerably up-regulated the Galectin-8 transcriptional level of the intestine，liver，head kidney，and spleen in hybrid grouper（P < 0.05），which suggested that FA served as an immunostimulant to improve its innate immunity. Nevertheless，Galectin-8 immune functions of the hybrid grouper are unclear，and therefore further studies are needed to illustrate its role in defense against pathogen invasion.

Key words: Epinephelus fuscoguttatus ♀ × E. polyphekadion ♂, Galectin-8, ferulic acid, gene cloning, expression analysis

付伟杰, 邝杰华, 罗君, 黄建盛, 陈有铭, 陈刚. 杉虎斑Galectin-8基因克隆及其在不同阿魏酸水平饲料下的表达响应[J]. 生物技术通报, 2022, 38(12): 312-323.

FU Wei-jie, KUANG Jie-hua, LUO Jun, HUANG Jian-sheng, CHEN You-ming, CHEN Gang. Gene Cloning of Galectin-8 in Epinephelus fuscoguttatus♀×E. polyphekadion♂ and Its Expression Responses Under Different of Ferulic Acid Level[J]. Biotechnology Bulletin, 2022, 38(12): 312-323.

图/表 9

表1 试验饲料组成及营养水平

Table 1 Ingredient composition and nutrient levels of the experimental diets

原料 Ingredient	比例Percentage/%	营养水平Nutrient level	比例Percentage/%
鱼粉Fish powder	45	水分Moisture	9.2
酪蛋白Casein	12	粗蛋白 Crude protein	51.30
小麦谷朊粉 Vital wheat gluten	8	粗脂肪 Crude lipid	10.4
玉米蛋白粉 Corn gluten meal	5	粗纤维 Crude fiber	0.1
面粉Wheat flour	17.22	粗灰分Ash	9.6
鱼油Fish oil	6
大豆卵磷脂Soybean lecithin	2
维生素与矿物质预混料 Vitamin and mineral premix^a	2
氯化胆碱Choline chloride	0.5
磷酸二氢钙 Ca（H₂PO₄）₂	1.5
维生素C Vitamin C	0.05
乙氧基喹啉Ethoxyquin	0.03
微晶纤维素Microcrystalline cellulose	0.4
诱食剂Attractant	0.1
FA预混料FA premix^b	0.2
Total	100

表2 本研究所用引物

Table 2 Primers used in this study

引物 Primer	序列Sequence（5'-3'）	用途 Application
Gal-8-F	TGATGCCGACAGGTTCCAGATAGA	中间片段验证 Middle sequence verification
Gal-8-R	GATGTTGACGCCCAGCAGTGT	中间片段验证 Middle sequence verification
UPM-long	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	RACE通用引物 Universal primers for RACE
UPM-short	CTAATACGACTCACTATAGGGC	RACE通用引物 Universal primers for RACE
Gal-8-5'GSP	TGCCCTCTGCGTTGGGATTG	5'端序列克隆 5' RACE
Gal-8-5'NGSP	GGAGGATGCCGACAGCCTGAATA	5'端序列克隆 5' RACE
Gal-8-3'GSP	GAGGTGAACTGGCTGGACTGAG	3'端序列克隆 3' RACE
Gal-8-3'NGSP	TCAGGGTTGCTGTCAATGGGCTC	3'端序列克隆 3' RACE
Gal-8-RT-F	AACACAGAGTGGACCTGGACCG	实时荧光定量PCR Quantitative real-time PCR
Gal-8-RT-R	CCAACACTCAGTCCTTTAGCCA	实时荧光定量PCR Quantitative real-time PCR
β-actin-F	CTCTCGGCTGTGGTGGTGAA	内参基因 Housekeeping gene
β-actin-R	CGTGATGGACTCTGGTGATGGT	内参基因 Housekeeping gene

图1 杉虎斑Galectin-8的全长cDNA核苷酸序列和推导的氨基酸序列下划线表示起始密码子；星号表示终止密码子；灰色阴影的氨基酸序列代表2个CRD结构域；黑色方框的氨基酸序列为CRD中的保守基序HXNPR与WGXEE（X表示任意氨基酸）；三角形代表预测的N-糖基化位点：圆表示预测的磷酸化位点；加粗斜体代表加尾信号

Fig. 1 Full length cDNA nucleotide and deduced amino acid sequences of Galectin-8 in hybrid grouper The initiation codon（ATG）is underlined. The stop codon is indicated by asterisks. The carbohydrate recognition domain is highlighted by shading. The conserved motifs in the CRD domain are boxed in black color. The triangle indicates the putative site for N-linked glycosylation. The circle indicates the putative site for phosphorylation. The polyadenylation signal sequence is shown in bold italics

图2 同源建模预测的Galectin-8蛋白三维结构

Fig. 2 Tertiary structure of Galectin-8 predicted by homol-ogy modeling

图3 杉虎斑与其他物种 Galectin-8氨基酸序列的多重比对 N端与C端的CRD分别用蓝色和绿色箭头线显示；红色方框代表CRDs中的保守基序；黑色方框表示连接肽

Fig. 3 Multiple sequence alignment of Galectin-8 amino acid sequences between hybrid grouper and other species The N-terminal and C- terminal CRDs are represented by blue and green color arrow headed lines，respectively. Conserved motifs in the CRD domain are boxed in red color；and the hinge sequences（linker peptide）of each ortholog are boxed in black color

表3 杉虎斑Galectin-8氨基酸序列多重比对与构建系统进化树所用到的氨基酸序列

Table 3 Amino acid sequences used for multiple sequence alignment and phylogenetic tree construction for Galectin-8 from hybrid grouper

物种Species	氨基酸Amino acids	一致性Identity/%	GenBank登录号GenBank accession number
杉虎斑 Epinephelus fuscoguttatus♀ × E.polyphekadion♂	319	—	OL826837
鞍带石斑鱼 E. lanceolatus	319	98.43	XP_033503883.1
许氏平鲉 Sebastes schlegelii	322	82.61	QJD13864.1
条石鲷 Oplegnathus fasciatus	313	79.31	ANN46245.1
蓝鳍金枪鱼 Thunnus maccoyii	313	75.94	XP_042246845.1
罗非鱼 Oreochromis niloticus	321	72.05	XP_003446682.2
?鱼 Echeneis naucrates	314	70.53	XP_029352421.1
大西洋鲑 Salmo salar	296	63.84	NP_001133778.1
香鱼 Plecoglossus altivelis	297	52.98	QNB48521.1
黄姑鱼 Nibea albiflora	313	47.48	KAG8013601.1
智人 Homo sapiens	317	54.09	AAF19370.1
北极熊Ursus maritimus	316	52.85	XP_008687860.1
牛 Bos taurus	357	48.44	NP_001039419.1
麻雀 Passer montanus	341	53.94	XP_039562765.1
原鸡 Gallus gallus	315	53.73	NP_001010843.1
白鹭 Egretta garzetta	317	52.85	KFP20361.1
虎斑响尾蛇 Crotalus tigris	297	49.36	XP_039208905.1
眼镜王蛇 Ophiophagus hannah	294	48.54	ETE69412.1
热带爪蟾 Xenopus tropicalis	315	49.69	NP_001135558.1
中华大蟾蜍 Bufo gargarizans	314	48.59	XP_044144965.1

图4 邻接法构建Galectin-8基因氨基酸序列的系统发育进化树杉虎斑在进化树上用“●”标示出来，距离标尺为0.1

Fig. 4 Phylogenetic tree of the amino sequences of Galectin-8 genes constructed by neighbour-joining method Hybrid grouper is marked with ‘●’ on the phylogenetic tree. The scale bar of 0.1 indicates genetic distance.

图5 杉虎斑Galectin-8在各组织的相对表达量数据以平均值±标准差表示，不同的小写字母代表差异显著（P < 0.05）；下同

Fig. 5 Relative expressions of Galectin-8 in the different tissues from hybrid grouper Values are displayed as mean ± SD；values that do not share the common lowercase letters differ significantly（P < 0.05）；the same below

图6 饲料阿魏酸不同添加水平对杉虎斑肝脏、肠道、头肾和脾脏的Galectin-8基因表达量的影响

Fig. 6 Effects of dietary supplementation of ferulic acid（FA）on the relative mRNA expressions of Galectin-8 in the liver，intestine，head kidney and spleen of hybrid grouper

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