Construction Of Full-length CDNA Library Of Hemocyte From Aeromonas Hydrophila Challenged Hyriopsis Schlegeli And Sequence Analysis Of Cyclophilin A Gene

Hyriopsis schlegelii originated in Biwa lake of Japan and was introduced to China in 1997. Compared with other freshwater pearl mussel, the Hyriopsis schlegelii has high rate of growth, better disease-resistance and pearling production. Considering economical interests, it is necessary to select breed with better disease-resistance phenotype.A full-length haemocytes cDNA library was constructed in order to clone genes involving in Aeromonas Hydrophila defenses from Hyriopsis Schlegeli.Inactivated Aeromonas Hydrophila were used to chalenge Hyriopsis Schlegeli (fourth instar). And then we extracted the total RNAs from haemocytes using TRIzol reagent. The"anchor first-strand cDNA"containing a symmetrical sfiI restriction enzyme sites (A&B) was synthesized by transcription of total RNA with the SMART technique.The SMART switching mechanism at 5'end of the RNA transcript technique was used to construct the cDNA library.The titer of the unamplified cDNA library was 4×106cfu/cm3 with a recombinantrate of 90% and that of amplified one was 3.55×109cfu/cm3. A total of 102 clones was selected to be sequenced and 59 were functional sequences.The shortest sequence was 270bp and longest one was 2153 bp.The average length is 608.6 bp. The results indicated that a high quality library was meet the needs for the consequence gene clone and expressed sequence tag analysis.Cyclophylin A(Cyp A,) was isolated from cDNA library by screening.The full length of the Hyriopsis Schlegeli Cyp A (HsCyp A)was 1229 bp, Sequence analysis showed that HsCyp A cDNA contained 52bp 5'untranslated region(UTR),682bp 3' UTR and 29 bp poly(A) tail.Which also includes an open reading frame (ORF) of 495 bp and encoded 164 amino acid residues,which doesn't exist signal peptides.It's molecular weight is 17.26 kDa and pI 8.9.We also analysised it's secondary structure and homology-modeled the HsCyp A. A phylogenetic tree showed that they might have similarities in biological functions evolving from the same ancient gene.