Cloning,Bioinformatics and Expression Characteristics Analysis of Galectin-3 Gene in Chinese Giant Salamander

doi:10.7668/hbnxb.20195469

Abstract

Abstract:

In order to identify the molecular biological characteristics and expression patterns of Galectin-3 gene in Chinese giant salamander before and after infection,the Galectin-3 gene was amplified by RT-PCR,and its physicochemical properties and protein structure were analyzed by bioinformatics.Real-time Quantitative PCR(qRT-PCR)was used to detect the expression of Galectin-3 in different tissues of Chinese giant salamanders before and after Aeromonas hydrophila.The results showed that the length of Galectin-3 gene CDS region was 351 bp,encoding 116 amino acids,the theoretical isoelectric point(pI)was 5.87,the relative molecular weight was 13.53 ku,and it was a stable hydrophilic protein located in the cytoplasm.Structural analysis showed that Galectin-3 protein contained a GLECT domain.The phylogenetic tree showed that the Galectin-3 amino acid sequence was closely related to Rhinatrema bivittatum.qRT-PCR results showed that Galectin-3 was expressed in all tested tissues of the salamander,and the relative expression level was the highest in the skin.The relative expression levels of Galectin-3 in the liver,muscle,intestines and kidneys of Chinese giant salamander increased significantly and reached the highest value at 12 h of infection with A.hydrophila.The relative expression levels of Galectin-3 in the spleen and skin of Chinese giant salamander increased significantly and reached the highest value at 48 h of infection with A.hydrophila.In conclusion,Galectin-3 gene was actively expressed in the early stage of salamander infected by foreign bacteria,suggesting that Galectin-3 gene may be involved in the immune regulation of salamander.

Key words: Andrias davidianus, Galectin-3, Bioinformatics analysis, Real-time Fluorescence Quantitative PCR, Aeromonas hydrophila

CLC Number:

S917水产生物学

SHANG Wencong, LI Qinrong, WU Feifan, SU Shuxian, LUO Jianlin, WANG Kaizhuo, WANG Hui, MENG Yonglu, YE Ting, LI Can. Cloning,Bioinformatics and Expression Characteristics Analysis of Galectin-3 Gene in Chinese Giant Salamander[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 231-238. doi: 10.7668/hbnxb.20195469.

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Figures/Tables 10

Tab.1 Primer information

引物名称 Primer names	引物序列(5'—3') Primer sequence(5'—3')	用途 Usage
Galectin-3-RT-F	CTTCTTCATAAAGCACC	Galectin-3片段扩增
Galectin-3-RT-R	TATTCCGCATTGGGTGAC
Galectin-3-CDS-RT-F	TGGCATCAGACCATCAAGAC	Galectin-3 CDS扩增
Galectin-3-CDS-RT-R	TCATAGGATTTTTATATTCAACG
β-actin-F	TGAACCCAAAAGCCAACCGAGAAAAGAT	内参基因
β-actin-R	TACGACCAGAGGCATACAGGGACAGGAC
Galectin-3-Q-F	ACTTGGCTAACGGAGTTGGA	实时荧光定量PCR
Galectin-3-Q-R	AATCCCCAGAGGCGGAAAAC

Fig.1 Agarose gel electrophoresis of PCR amplified products of Galectin-3 gene in Andrias davidianus M.DL2000 DNA Marker;1.Full length amplification product; 2.CDS amplification products.

Fig.2 Deduced amino acid sequence of the giant salamander Galectin-3 Coded amino acid sequence of A.davidianus Galectin-3 gene with start and stop codons in red.

Fig.3 Hydrophobic/hydrophilic analysis of Galectin-3-encoded proteins in the giant salamander

Fig.4 Prediction of conserved domain of A.davidianus Galectin-3 protein

Fig.5 Phosphorylation site analysis of Galectin-3 amino acid sequence in the giant salamander

Fig 6 Secondary structure prediction(A)and tertiary structure prediction(B)of A.davidianus Galectin-3 protein A.Secondary structure prediction:Yellow.Random coil, Purple.Extended strand,Blue.α-helix;B.Tertiary structure prediction.

Fig 7 Galectin-3 protein phylogenetic tree

Fig.8 Expression levels of Galectin-3 in different tissues of healthy giant salamander Different lowercase letters indicate significant differences between different tissues(P<0.05).

Fig.9 The expression of Galectin-3 in different tissues of A.davidianus after infection Different capital letters indicate significant differences(P<0.05)between different treatment groups at the same time point,and different lowercase letters indicate significant difference(P<0.05)between the same treatment groups at different time points.

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