cDNA Cloning and Study on Tissue Distribution of F0-ATP Synthase b-chain of Litopenaeus vannamei

Abstract:

White spot syndrome virus (WSSV) is a major fatal pathogen to shrimp. It is known that the b-chain of F0-ATP synthase plays a key role in the synthesis of ATP in all living organisms. Evidence from our previous research indicated that the b-chain of F0-ATP synthase of Litopenaeus vannamei was involved in WSSV infection. However the full-length sequence of the b-chain of F0-ATP synthase in L. vannamei has not been available yet. In this study we cloned the full cDNA using reverse transcription PCR (RT-PCR) and the rapid amplification of cDNA ends (RACE) method. Bioinformatics analysis was performed to predict the amino acid sequence and the secondary and space structure of the b-chain of F0-ATP synthase. We also mapped the homology and phylogenic tree using ClustalX 1.83 and MEGA 4.02. Immuno-histochemical and flow cytometry analysis were carried out to detect the tissue distribution of the b-chain of F0-ATP synthase in L. vannamei. The results showed that the 1129 bp full length cDNA was successfully cloned. Bioinformatics analysis indicated that the full length cDNA had an open reading frame (ORF) of 744 bp that encoded 248 amino acids, and that the predicted molecular weight of the mature peptide was 28.2 kDa. The homology analysis of the b-chain of F0-ATP synthase between species demonstrated that there was a higher similarity between L. vannamei and Caligus clemensi (50%), and Drosophila melanogaster (60%). Immuno-histochemical results showed that the b-chain of F0-ATP synthase was widely distributed in the gill of L. vannamei. Flow cytometry analysis indicated that the b-chain of F0-ATP synthase was distributed on the surface of hemocytes as well as in the cytoplasm of L. vannamei. These results suggested that the b-chain of F0-ATP synthase could be located on the surface as well as in the cytoplasm of hemocytes when mediating the WSSV infection in L. vannamei. Our study shed lights on the further understanding of the role of the b-chain of F0-ATP synthase in WSSV infection.