Identification and cloning of the alpha2-macroglobulin of giant freshwater prawn Macrobrachium rosenbergii and its expression in relation with the molt stage and bacteria injection.

The full-length complementary (c)DNA of the alpha2-macroglobulin (alpha2M) gene was cloned from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The 4875-bp cDNA contains an open reading frame (ORF) of 4419 bp, a 95-bp 5'-untranslated region (UTR), and a 361-bp 3' UTR containing the poly A tail. The ORF encodes a protein of 1472 amino acids (aa) with a 23-residue signal sequence. The molecular mass of the deduced amino acid sequence (1449 aa) was 163.29 kDa with an estimated pI of 4.88. The M. rosenbergii alpha2M sequence contains putative functional domains including a bait region and a GCGEQ internal thiol ester site, and a receptor-binding domain is present as in other aquatic arthropod alpha2Ms. Sequence comparison showed that alpha2M of this prawn had overall respective identities of 38.4%, 45.9%, 45.9%, and 46.0% to those of Scylla serrata, Litopenaeus vannamei, Penaeus monodon, and Marsupenaeus japonicus. A phylogenetic analysis revealed that M. rosenbergii alpha2M is the more-primitive genotype, and it showed significant differentiation from marine crustacean alpha2Ms. alpha2M was mainly expressed in haemocytes. The quantitative real-time RT-PCR analysis showed that alpha2M mRNA transcripts significantly increased in the A stage, achieved the highest level in the C stage, then declined in the D(0/1) stage, and reached the lowest level in the D(2/3) stage in haemocytes of prawn. alpha2M's expression in haemocytes of M. rosenbergii significantly increased at 24 h and 12 h after injection with heat-killed Lactococcus garvieae and Vibrio alginolyticus, respectively, which indicates that alpha2M is involved in the immune response of prawn.