Molecular Cloning, Recombinant Expression Analysis Of Phospholipase A₂ And Pore Forming Protein Genes From Rhopilema Esculentum

Rhopilema esculentum belongs to the C nidaria, Scyphozoa, Rhizostomeae, Rhizostomatidae and Rhopilema. As a important edible commodity, Rhopilema Esculentum plays an irreplaceable role in china’s marine fisheries. Rhopilema esculentum is a ideal model organisms to study the regulation of gene expression during jellyfish development and the components, structure and function of jellyfish toxin, in which the basic biological research is exhaustive while it’s culture and enhancement technology is mature.On the base of 454 transcriptome sequencing, we cloned two phospholipase A2 c DNA, named Re PLA2-1 and Re PLA2-2, two MACPF domain contain pore forming protein, named Re PFP-1 and Re PFP-2 from Rhopilema esculentum by expressed sequence tag（EST） and rapid amplification of c DNA ends（RACE） techniques. This research includes the following contents:The full- length c DNA sequence of Re PLA2-1、Re PLA2-2 was obtained by rapid amplification of c DNA ends techniques. The full- length c DNA of Re PLA2-1consisted of 824 nucleotides with a 5’UTR of 48 nucleotides, a 3’UTR of 272 nucleotides and a Open reading frame（O RF）of 504 nucleotides. The ORF encoding a polypeptide of 167 amino acids, with a theoretical mass of 18.93 k Da and a p I of 8.02. The full- length c DNA of Re PLA2-2consisted of 840 nucleotides with a 5’UTR of at least 6 nucleotides, a 3’UTR of 336 nucleotides and a Open reading frame（ORF）of 495 nucleotides. The ORF encoding a polypeptide of 165 amino acids, with a theoretical mass of 18.747 k Da and a p I of 8.94. The genomic structure of Re PLA2-1、Re PLA2-2 was obtained by overlap PCR techniques. The Re PLA2-1 gene was 2937 bp in length, contains four exons, with coding regions of 39 and 388 nt, intervened by three introns. Re PLA2-2 gene was 2113 bp in length, contains two exons, with coding regions of 189 and 324 nt, intervened by one intron of 1273 nt. All the introns-exon junctions that are typical donor and acceptor splice sites have followed the GT/AG rule, in which the introns begin with GT and end with AG. The results of blastp reveal that the Re PLA2-1、Re PLA2-2 are the members of Phospholip_A2₃ superfamily. Phylogenetic analysis suggests that Re PLA2-1、Re PLA2-2 share common evolutionary origin with C. magus、N. vectensis、C. gigas. The distribution of Re PLA2-1、Re PLA2-2 transcripts and Protein in differe nt development stages of Rhopilema esculentum were examined by RT-PCR. Re PLA2-1、Re PLA2-2 had a constitutive expression in all four examined stages, and Re PLA2-1 had a significantly high expression in strobila and followed by scyphistoma stage, while a lower level of transcription was detected in medusa, ephyra. The expression levels in strobila, scyphistoma and medusa were 12.2-fold, 7.6-fold and 5.5-fold higher than that in ephyra, respectively. While Re PLA2-2 had a significantly high expression in medusa and followed by ephyra stage, while a lower level of transcription was detected in scyphistoma, strobila. The expression levels in medusa, ephyra and scyphistoma were 10.8-fold, 4.4-fold and 1.3-fold higher than that in strobila, respectively. Recombinant expression of the mature peptide of Re PLA2 was successfully performed, and the recombinant protein expressied in inclusion bodies after inducted by IPTG. A hydrolyze activity was detected from the refolding recombinant protein.The full- length c DN A of Re PFP-1 consisted of 2303 nucleotides with a 5’UTR, a 3’UTR, a Open reading frame（ORF）of 1883 nucleotides and encoding a polypeptide of 610 amino acids. The 3’UTR contained a canonical polyadenylation signal sequence AATAAA and a poly（A） tail. The full- length c DNA of Re PFP-2 consisted of 2212 nucleotides with a 5’UTR, a 3’UTR, a Open reading frame（ORF）of 1842 nucleotides encoding a polypeptide of 613 amino acids. The 3’UTR contained a canonical polyadenylation signal sequence AATAAA and a poly（A） tail. The amino acid sequence of Re PFP-1show 65% identical to that of PFP-2, and the two Re PFPs all contain MACPF domain which are found in all MACPF domain-containing proteins, but a unusual vitelline membrane outer layer proteins I was present in the C-terminal of all the two PFPs we cloned. The homology and phylogenetic analysis revealed that the deduced amino acid sequence of Re PFP-1 and Re PFP-2 gene exhibited the highest identity with those of Pomatoceros lamarckii, Mytilus galloprovincialis and Tetrahymena thermophila, and formed a single lineage from the other MACPF superfamily members. The Re PFP m RN A levels at R.esculentum developmental stages were detected at scyphistoma, strobila ephyra and medusa stages. The highest level was observed in the scyphistoma stage. Thus, we speculate the role of Re PFP-1 and Re PFP-2 protein is not acting as a toxin, but participation in the regulation of early development of jellyfish. Primers were designed based on the full- length c DNA sequence of Re PFP-1 and Re PFP-2 from R.esculentum to clone the MACPF domains of Re PFP-1 and Re PFP-2 genes. The MACPF domains fragments were subcloned into the prokaryotic expression vector p ET-28 a to successfully construct recombinant plasmid pet28-Re PFP-1and pet28-Re PFP-2. Then the recombinant plasmid was transformed into E. coli BL21（DE3） cell and recombinant expression induced by IPTG, but both the two recombinant Re PFPs did not show any antibacterial activity in our study.