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| Molecular Cloning and mRNA Expression Analysis of Calcineurin B Homologous Protein 2 Gene in Black Porgy Acanthopagrus schlegelii |
| CHEN Shuyin, ZHANG Zhiyong, SUN Ruijian, JIA Chaofeng, MENG Qian, ZHU Fei, ZHANG Zhiwei, WU Guojun |
| Marine Fisheries Research Institute of Jiangsu Province, Nantong 226007, China |
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Abstract Full length cDNA sequence of calcineurin B homologous protein 2 (CHP2) gene as a key molecule involved in regulation of intracellular pH and cell proliferation was amplified in black porgy Acanthopagrus schlegelii (AsCHP2) by RACE PCR, and the sequence characteristics and homology of the AsCHP2 were analyzed by bioinformatics. The expression of AsCHP2 mRNA was detected in whole black porgy larvae, and brain, heart, muscle, liver and gonads of 90 d or 180 d old black porgy hybrid F2 (red sea bream Pagrus major ♂×black porgy ♀) and, backcross F1 (black porgy♂×hybrid F1♀) by real-time fluorescent quantitative PCR. It was found that the AsCHP2 cDNA was 1722 bp in length, including a 69 bp 5′-untranslated region (UTR), a 1069 bp 3′-UTR and a 585 bp open reading frame encoding a peptide of 194 amino acids. The protein contained two conserved EF-hand domains and a nuclear export sequence, the molecular weight of about 21. 87 ku, pI of about 4. 89. Secondary and three-dimensional structure of AsCHP2 was developed by homology modeling. Nuclear sequence alignment revealed that AsCHP2 shared higher identities (86%—81%) with other fishes, and the alignment of the predicted proteins sequence of AsCHP2 had 80%—72% of similarity with other fishes. The evolutionary relationships of AsCHP2 amino acid sequences with other kinds were analyzed using the methods of NJ tree and ML tree. Sequence alignment and structure comparison revealed that AsCHP2 were members of the conservative CHP2. The AsCHP2 appeared to be constitutively expressed in early larval development (5—35 d) and the 90-day and 180-day old individuals, and had high expression level in all tissues from the individuals of 90-day old. CHP2 mRNAs was ubiquitously expressed in all tested tissues, and far more abundant in the heart, as about several times as that in gonad in black porgy, the hybrid F2 and the backcross F1. These findings suggest that the CHP2 gene might be involved in the growth and development of sea fishes.
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Received: 17 April 2019
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