THE FULL-LENGTH cDNA LIBRARY OF HEMOCYTE INDUCED BY AEROMONAS HYDROPHILA AND MOLECULAR CHARACTERISTICS OF CYCLOPHILIN A FROM HYRIOPSIS SCHLEGELII

Hyriopsis schlegelii, originated from the Lake BIWA of Japan, was introduced into China in 1997. In order to seek for genes related to the freshwater mussel on immune system, a full-length haemocyts cDNA library was constructed by using SMART (switching mechanism at 5 end of the RNA transcript) technique. The total RNA was isolated from the four years old mussel blood hemocyte induced by Aeromonas hydrophila for 14 hours. The anchor first-strand cDNA containing a sites (A B) of symmetrical sfi I restriction enzyme was synthesized by reverse transcription, and the double-strand cDNA was synthesized and amplified by LD-PCR (long-distance PCR). After digested by the proteinase K and sfi I restriction enzyme, size fractionation by CHROMA SPIN-400 columns, ligated with the sfi I digested pDNR-LIB vector and transformed into E. coli DH10B by thermal shock, the full-length cDNA library was constructed. It was the first time to construct a full-length cDNA library from blood hemocyte of the freshwater mussel. The titer of the primary constructed cDNA library was 4106 cfu/cm3 with a high recombination rate of 90% and the amplified one was 3.55109 cfu/cm3. 672 clones were sequenced randomly selected from the library and 436 have the homologous gene information after analyzed by BLASTx in the website of NCBI. Among the sequences, the shortest one was 270 bp and the longest one was 2153 bp, and average length was 608 bp. Results indicated it was a high quality cDNA library. The full length of the Cyp A (HsCyp A), which may provide immune stress function in some species, was isolated from the cDNA library by screening of H. schlegelii. It has 1229 base pair (bp), containing a 52 bp 5 untranslated region (UTR), a 495 bp open reading frame, a 682 bp 3-untranslated sequence, and a 29 nucleotide long poly (A) tail. The initiator codon (ATG) and the stop codon (TAA) can be found at the position of 53 and 545, but can not find the putative polyadenylation signal (AATAAA). The predicted protein sequence consisted of 146 amino acids with a calculated molecular mass of 17.26 kD and an isoelectric point of 8.9. The secondary structure, 3D homology-modeled structure and phylogenetic analysis indicated the HsCyp A was extremely conservative during the evolutionary history. All the results showed that the Cyp A was not only a consistent protein, but also may play important roles in the immune system the lower aquatic animal. This work may provide important information about specific genes for mussel immune, and also provide some background for future research about the immune mechanisms of Cyp A in freshwater mussel.