Identification and cloning of the α2-macroglobulin of giant freshwater prawn Macrobrachium rosenbergii and its expression in relation with the molt stage and bacteria injection

Abstract

The full-length complementary (c)DNA of the α2-macroglobulin (α2M) 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 p I of 4.88. The M. rosenbergii α2M 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 α2Ms. Sequence comparison showed that α2M 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 α2M is the more-primitive genotype, and it showed significant differentiation from marine crustacean α2Ms. α2M was mainly expressed in haemocytes. The quantitative real-time RT-PCR analysis showed that α2M 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. α2M'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 α2M is involved in the immune response of prawn.