Construction and Ests Analysis of the Subtraction Cdna Library for Larvae of Lutjanus Sanguineus Induced by Vibrio Alginolyticus
|School||Guangdong Ocean University|
|Keywords||Vibrio alginolyticus suppression subtractive hybridization expressed sequence tags(ESTs) Lutjanus sanguineus|
According to the previous work, we have demonstrated the maternal antibody of Lutjanus sanguineus would be fully degraded in 7 days post hatch when the specific immune response had not been developed at that time. To evaluate the immune response of the larvae will benefit for the research on improving the survival rate during the early stages in mariculture for L. sanguineus. As a result, we construct the subtraction cDNA library of the larvae fish of L. sanguineus induced by the bacteria pathogen, Vibrio alginolyticus by suppression subtractive hybridization (SSH) method. Furthermore, the immune-related ESTs were delineated by bioinformatics analysis.The tester cDNA was obtained from the larvae of Lutjanus sanguineus induced by vibrio alginolyticus(1.0×10~8 cfu/mL), the driver cDNA was obtained from the larvae induced by PBS, the cDNA library was constructed by suppression subtractive hybridization with tester cDNA and driver cDNA. The results showed that the primary cDNA library contained 2×10~6 clones, PCR results showed that the inserts varied from 0.3 kb to 1.0kb, and the recombination rate was about 90%. A total of 1000 clones in the SSH library were randomly selected and sequenced 910 clones were sequenced successful. Based on BLAST searches in NCBI , the 720 ESTs had significant matches to known sequences in the GenBank, 140 ESTs had matches to known sequences in the GenBank, 50 ESTs had no significant matches to known sequences in the GenBank. The sequences of these ESTs were subjected to GO annotation. According to their physioligical function, they can be subdivided into 8 categories: Cellimmunity, defense, stress(7ESTs, 1%), Cytoskeleton (34ESTs, 4%), Ribosomal proteins(258ESTs, 29%), Cell metabolism, respiratory chain (430ESTs, 51%), Cell signaling, adhering (43ESTs, 5%), Cell cycle, DNA replication, protein regulation, transcription, translation(25ESTs, 3%), transferproteins(17ESTs, 2%), others proteins (43ESTs, 5%).Altogether, the results obtained from this study provide valuable information on further investigate the molecular immune mechanism of larvae of Lutjanus sanguineus. Furthermore, the discovery and expression study of genes related to immune defense in this study will facilitate studies in genetic breeding and genetic improvement with the aim to enhance disease resistance of larvae.