Cloning, sequencing and analysis of housekeeping genes towards the development of molecular stress response markers in seagrasses
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Seagrass meadows are important primary producers and habitats in estuaries and near-shore marine environments, but many populations are in decline due to anthropogenic influences. Measurements of biomass are commonly used to gauge the physiological status of seagrass meadows, but these are "lagging indicators" of the underlying causal event(s) and have not fully answered questions about why, despite attempts to correlate with environmental conditions. The goal is to develop a method for transcriptomic measurements to compare relative long-term stress response levels between impacted and nonimpacted seagrasses. Given the lack of genomic information for seagrasses in the western Gulf of Mexico, it was first necessary to obtain seagrass genomic sequences based on knowledge of model systems. Control (Act1, Gapdh) and stress genes (Apx1, non-symbiotic Hb1, and Pal1) were first identified by literature search using the rice (Oryza sativa) genome as a model. Multiple alignments were performed to identify conserved regions and design degenerate PCR primers used for cloning and sequencing from five seagrass species: Halodule beaudettei (synonymous with H. wrightii, Cymodoceaceae), Cymodocea filiformis (Cymodoceaceae), Thalassia testudinum (Hydrocharitaceae), Halophila engelmannii (Hydrocharitaceae), and Ruppia maritima (Ruppiaceae). Amplification of the desired stress-related genes from seagrasses was unsuccessful. Hb1 primers yielded PCR products from H. beaudettei around the expected size (~759 bp), but sequence analysis identified this as a bacterial-like NAD/NADP octopine/nopaline dehydrogenase. Using genomic DNA, actin gene fragments (1-1.8 kb) corresponding to exons 2-4 were amplified from five species, and Gapdh (exons 5-9) was amplified from H. beaudettei. Intron length varied for actin with C. filiformis containing the largest introns. Splicing junctions were verified comparing cDNA sequences from H. beaudettei. Actin and GAPDH sequences were aligned in MEGA using MUSCLE and compared with other plant sequences in GenBank®. A phylogenetic tree was constructed for each gene using Maximum Likelihood with 1,000 bootstrap replicates. Actin cDNA sequences from the same families grouped together to form clades reaffirming phylogeny. However, the genomic sequences of H. beaudettei actin, as well as GAPDH, did not group together with the expected clade, unlike the cDNA sequences from the same species. The genomic actin sequences were most closely related to rice Act1, which is grouped with reproductive actins in other plants. Similarly, the genomic sequences of GAPDH did not group together with the mRNA sequences, but instead grouped with dicots reaffirming BLAST search results. Mean codon bias differences in genomic sequences vs. cDNA along with differences in theoretical isoeletric points seem to indicate multiple members of gene families for actin and GAPDH in H. beaudettei, similar to previous work in all angiosperms studied thus far. This finding suggests that the genomic vs. cDNA clones of both actin and GAPDH may represent differentially expressed paralogs. This work raises the interesting possibility that expression patterns of individual housekeeping paralogs could be used as stress indicators. Future work should include high-throughput sequencing to analyze expressed housekeeping genes under a variety of environmental conditions and to identify stress-related gene candidates in the transcriptome.