Construction And Analysis Of The Subtracted CDNA Library From Hyriopsis Cumingii Liver

Hyriopsis cumingii is one of the most important endemic freshwater pearl species in China. H. cumingii Plague is an infectious disease and spreads fast. It severely affects the production of freshwater pearl mussels by causing high mortality with no affective treatments. Studies on H.cumingii plague pathogen, including pathogenic bacteriology and virology, has reported since 1980s. In this study, mussel samples with typical symptoms of H. cumingii plague were collected in September 2005. To establish technology and methodology of genetic breeding resistant to H.cumingii plagues, the newly developed technique of suppression subtractive hybridization (SSH) was used to construct a cDNA library of the H.cumingii liver and screened for immune related factors associated with H.cumingii plagues.1. Construction of subtracted liver cDNA library in Hyriopsis cumingiiSubtracted liver cDNA library of healthy and disease-resistant H.cumingii (survivorsafter infected) were constructed using the suppression subtractive hybridization technology (SSH). 214 out of the 300 positive cDNA clones randomly selected for sequencing were effective in obtaining of cDNA sequences.2. Sequencing analysis of positive clones in Hyriopsis cumingiiSequence homology search showed that 120 out of the 214 effective cDNA sequences were homologous with high sequence identities to known sequences of functional proteins in the GenBank (E value≥6). Sequences of 30 positive clones had high similarities with open reading frame (ORF) of molecules of unknown function referred as hypothetical protein in the database. 21 sequence showed no significant similarity to any other sequences, which might be a novel EST sequence and a potential 5'- or 3'- UTR sequence in the genome that had been reported previously. 21 known function EST for 40 different genes were reported in H.cumingii.for the first time in this study.According to the classification of functional genes by Adam, the identified EST in H.cumingii fell into eight categories relevant to cell division, cellular structure and movement, metabolism, signal transduction, cell immune-action, gene and protein expression associates and other proteins with unknown functions. 40sequences, 18.2% of the total analyzed clones, reported in this study are related to the immune factors of cell recovery.