Gene Cloning of Galectin-8 in Epinephelus fuscoguttatus♀×E. polyphekadion♂ and Its Expression Responses Under Different of Ferulic Acid Level

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

Abstract

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

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.

Figures/Tables 9

References 46

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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

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

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

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

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