Cloning, Tissue Expression and Primary Functional Analysis of PTEN Gene in Largemouth Bass (Micropterus salmoides)
LI Jia-Xin1, GAO Feng-Ying2,*, DONG Jun-Jian2, SUN Cheng-Fei2, ZHANG He-Tong2, ZHU Zhi-Lin1, WANG Qing-Kui1,*
1 Fisheries College, Tianjin Agricultural University, Tianjin 300380, China; 2 Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences/Key Laboratory of Tropical and Subtropical Aquatic Resources Utilization and Breeding, Ministry of Agriculture and Rural Affairs, Guangzhou 510380, China
Abstract：Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is the first tumor suppressor gene with bispecific phosphatase activity, which plays an important role in many signaling pathways. To clarify the regulatory role of PTEN in nuclear factor kappa-B (NF-κB), interferon (IFN) and activator protein-1 (AP-1) signaling pathways, which are closely related to the immune response in fish, in this study, the ORF region of PTEN gene was cloned from largemouth bass (Micropterus salmoides), the effects of pathogen-associated molecular patterns Nocardia seriolae, Largemouth bass virus (LMBV), Poly I:C challenge on PTEN gene expression in largemouth bass were detected by qPCR in vivo. To investigate the subcellular localization of PTEN, eukaryotic expression plasmid pCMV-Flag-PTEN was constructed, and the effect of PTEN overexpression on NF-κB, AP-1 and IFN promoter activation was investigated. Protein sequence analysis showed that PTEN encoded a 439-amino acid protein, including a PTPc domain and a PTEN_C2 domain. In the phylogenetic tree, the PTEN protein of largemouth bass was clustered with other fish. The results of qPCR showed that PTEN was widely expressed in many tissues, and highly expressed in spleen and gill. After LMBV and Poly I:C stimulation, the expression of PTEN decreased first and then increased or increased first and then decreased. The results of subcellular localization showed that PTEN was distributed in the cytoplasm and nucleus of HEK-293T cells. Double luciferase activity assay showed that overexpression of PTEN could enhance AP-1 activation and inhibit the activity of NF-κB and IFN promoter. These results suggested that PTEN played an important regulatory role in the innate immune response against bacteria and viruses by activating AP-1 and inhibiting NF-κB and IFN signaling pathways. This study provides basic data for the clarify of immune response mechanism of largemouth bass against disease, and provides theoretical basis for the development of antibacterial vaccine and the breeding of disease-resistant varieties (lines).
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