Molecular Cloning And Characterization Of A CDNA Encoding Extracellular Signal-regulated Kinase(ERK) From Blood Clam Tegillarca Granosa

The blood clam Tegillarca granosa is a shellfish with high economic value in the Asia-Pacific region.T.granosa grows in an aquatic environment with kinds of pathogens which are the major cause of diseases in T.granosa.In the last ten years,the production of T.granosa has been continually declining due to multiple pathogenic microorganism,which affects the development of the aquaculture on T.granosa.T.granosa entirely depends on innate immunity for pathogen defense.However,there are very few reports about immune response of T.granosa to various pathogens.The mitogen-activated protein kinase(MAPK)cascade is one of the most important signaling mechanisms in response to environmental stimuli.ERK,as an important member of MAPK,takes part in lots of cellular and physiological processes,which plays a vital role in the life.The regulating of ERK activity is important for cell proliferation to occur,ERK1 and ERK2 provide ERK activity in mammals and most vertebrates.There is only an overall 17% difference between the amino acid sequences of ERK1 and ERK2.One obvious difference between them is that ERK2 protein molecular weight is significantly larger than ERK1 protein in mammals.Only one ERK was reported in all invertebrates to date.In our study,we also found that T.granosa had only one ERK homolog.Phylogenetic tree analysis revealed that Tg ERK was closest to its homolog from the Haliotis discus discus.This is similar to the study of Drosophila melanogaster.In D.melanogaster,it was named rolled(for gene)or Rolled(for protein)and involved in several important cellular activities.In order to study how the ERK gene regulates immunity in blood clam when there is only one ERK,we used rapid amplification of cDNA ends method to clone and characterize the ERK homolog from T.granosa,which was designed as Tg ERK.The full-length cDNA sequence of TgERK was 1,644 bp,encoding a conserved S_TKc domain(residues 21-309)in the N-terminus.Tg ERK m RNA was expressed universally in all examined tissues with the highest expression level found in hemocytes.Lipopolysaccharide(LPS)and Vibrio alginolyticus challenges strongly enhanced expression of ERK in T.granosa,which was consistent with the in vitro challenge study in cultured T.granosa hemocytes.Pathogen invasion also upregulated expression of downstream genes in ERK signaling pathway,such as CREB,c-Fos and SIRT1.Moreover,Tg ERK knockdown resulted in the decreased expression of these downstream genes.Inhibition of ERK by its inhibitor U0126 inhibited T.granosa hemocytes viability in a dose-dependent manner.Taken together,our study demonstrated that Tg ERK was a crucial regulator of immune response to pathogen invasion,which indicated a new comprehension of hemocyte immunity in T.granosa and provided a novel key molecule in immune regulation for controlling diseases in T.granosa aquaculture.