Meeting Abstract

P3.23  Friday, Jan. 6  Identification of splice variants and protein expression levels for two Late Embryogenesis Abundant proteins in embryos of Artemia franciscana BOSWELL, L/C*; MOORE, D/S; HAND, S/C; Louisiana State University; Louisiana State University; Louisiana State University

Late Embryogenesis Abundant (LEA) proteins are highly hydrophilic, low complexity proteins that were originally discovered in plants. The expression of LEA proteins has been tightly correlated with desiccation tolerance in anhydrobiotic organisms. We have currently identified and sequenced six LEA genes in embryos of the brine shrimp Artemia franciscana. Two of the six Afrlea genes (Afrlea2 and Afrlea3m) have been cloned into a bacterial expression system, over-expressed, and purified. Based on deduced amino acid sequences, AfrLEA3m is predicted by subcellular targeting programs to be mitochondrial localized (now confirmed experimentally), while AfrLEA2 is predicted to be cytosolic. Multiple splice variants for both AfrLEA2 and AfrEA3m have been detected in A. franciscana using antibodies produced against recombinant AfrLEA2 and AfrLEA3m. Two splice variants are observed for AfrLEA2 with estimated molecular weights of 75 and 45 kDa, and four splice variants are observed for AfrLEA3m with estimated molecular weights of 29, 35, 45, and 52 kDa. The four splice variants detected for AfrLEA3m have been verified with LC tandem mass spectrometry. We have previously reported the mRNA expression of Afrlea2 and Afrlea3m to be several fold higher in two embryonic stages of A. franciscana that possess desiccation tolerance, when compared to the desiccation-intolerant nauplius larva. Furthermore we have now shown that protein expression for each of these two AfrLEA proteins is highest in diapause embryos and decreases throughout development to undetectable levels in 24 h nauplius larvae. We have also quantified the endogenous AfrLEA2 protein concentration of the 75 kDa splice variant to be approximately 0.9 mg per g embryo water for early post-diapause embryos. (Supported by NSF grant IOS-0920254)