Computational protocol: A novel and stress adaptive alternative oxidase derived from alternative splicing of duplicated exon in oyster Crassostrea virginica

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Protocol publication

[…] CvAOX mRNA sequences were obtained by searching Crassostrea gigas AOX (CgAOX) cDNA sequence (GenBank: ACL31211) in published and unpublished transcriptome databases of C. virginica. CvAOX sequences were aligned using Clustal W which revealed two variants differed by ~170 bp (Fig. ).To verify if the size difference is due to an indel in genomic DNA or from alternative splicing, primers (Table , Fig. ) were designed to amplify the region in question using both cDNA and genomic DNA as templates. Genomic DNA and cDNA from gills of nine C. virginica as well as genomic DNA of 31 oysters from four wild populations: Rhode Island (8), Delaware Bay (8), Florida (7) and Texas (8), were extracted and used for amplification. Polymerase Chain Reaction (PCR) was carried out in 10 µl volume containing 20 ng of DNA, 1 × PCR buffer, 1.5 mM of MgCl2, 0.2 mM of dNTP, 200 nM of each primer and 0.2 U of GoTaq polymerase (all from Promega) using the following profile: 95 °C for 5 min; 35 cycles of 95 °C for 30 sec, 57 °C for 30 sec, and 72 °C for 45 sec; and a final extension at 72 °C for 10 min. PCR products were analyzed in 1% agarose gels. To determine gene intron-exon structure, primers were designed to amplify genomic sequence of CvAOX based on cDNA sequences (Table ). PCR was carried out as described above using genomic DNA and the following profile: 95 °C for 5 min; 35 cycles of 95 °C for 45 sec, 56 °C for 45 sec, and 72 °C for 90 sec; and a final extension at 72 °C for 10 min. PCR products were purified and sequenced at GenScript Inc., USA. Sequences were assembled using SeqMan (DNASTAR Inc. http://www.dnastar.com). Introns were identified by sequence alignment and the GT-AG rule. Ten protein sequences of AOX representing Protista, Fungi, Plantae, and Animalia were obtained from NCBI (Table ) and aligned with the protein sequences of CvAOX and CgAOX. Active and functional sites in the protein sequence were determined according to Shiba et al. 2013. Phylogenetic tree was constructed with Bayesian model using the Markov Chain Monte Carlo (MCMC) approach. The parameters for MCMC sampling were: generations = 2,000,000 (standard deviation of split frequencies was < 0.01) with sample frequency = 1000, chains = 4, and burn-in value = 250 (corresponding to 25% of the samples). The method was implemented in MrBayes 3.1. […]

Pipeline specifications

Software tools Clustal W, MrBayes
Application Phylogenetics
Organisms Crassostrea virginica
Chemicals Oxygen