Construction And Analysis Of Liver Suppression Subtractive Hybridization Library Of Silver Carp (Hypophthalmichthys Molitrix) Exposed With Microcystin-LR And Cloning Partial Differentially Expressed Genes

With the aggressivation of eutrophication in water body, the harm of toxic cyanobacteria bloom on water environment and bio-safety leaded to increasingly concern worldwide. The cyanotoxins produced by blooms pose a great threat to the health of humans and aquatic animals. Microcystin-LR (MC-LR), which has the strongest toxicity, is frequently encountered and causes the most damages.Freshwater phytoplanktivorous fishes, such as silver carp (Hypophthalmichthys molitrix), consume great quantities of toxic blue-green algae (cyanobacteria), which may contain high concentrations of MCs. Compared with mammals, fish (especially phytoplanktivorous species) are more resistant to the toxic effects of MC-LR. But the molecular mechanisms of detoxification are not fully elucidated. Therefore, studying on the change of gene expression after exposed to MC-LR in silver carp has important theoretical and practical significance on mechanism of detoxification. In the present study suppression subtractive hybridization (SSH) was performed to construct the silver carp liver cDNA libraries exposed to MC-LR. In order to elucidate the detoxification mechanism at the molecular level, global gene expression profile was examined after functional annotation and category for qualified expressed sequence tags (ESTs) in SSH cDNA library, and some detoxification related genes were cloned. Several aspects included in the research were as followed:1. Silver carp liver exposure experiment and quantitation of the GST expression after exposure to MC-LRSilver carp were intraperitoneally injected with a single dose 200μg kg-1 body weight [bwt] of pure MC-LR dissolved in 0.8% sterile saline, Control fish received 0.8% sterile saline only. At 1, 3, 5 and 10 h post-injection, the liver from exposed group and control group fish were dissected out. The relative abundance of GST mRNA in silver carp livers was determined by RT-PCR, using beta-actin as the external control. Results showed that relative mRNA transcription levels of the GST gene in the silver carp liver significantly decilned at 1 h after expoed to MC-LR compared with the control group, which implies that the GST gene had participated in detoxification of MC-LR at 1 h post-injection. Hence, the liver cDNAs from 1 h post-injection fish (and the appropriate control group) were chosen for SSH.2. Construction and analysis of liver suppression subtractive hybridization library of silver carp (Hypophthalmichthys molitrix) exposed with MC-LRThe SSH cDNA library was successfully constructed with silver carp liver exposed with MC-LR. The forward and reverse subtracted libraries consisted of 2248 and 1686 positive clones respectively. Among the recombinant clones, 150 (70 forward and 80 reverse) were sequenced, then 88 ESTs (48 forward and 40 reverse) was obtained. These search results revealed that of the 88 positive clones, 75 represented unique genes, while 13 were duplicates.Among the 75 unique genes, 38 shared high homology with fish genes of known function, 11 were homologous to unknown genes or cDNA clones, while the remaining 26 were novel ESTs. The results of Blast comparisons of the subtracted ESTs were classified according to various putative cellular functions. The present study shows that many immune-related genes, transporters and some involved in cell metabolism were greatly altered. They would be play an important role in the detoxification process.3. Semi-quantitative RT-PCR studies on differentially expressed genes in liver of silver carpThree ESTs from forward and reverse libraries respectively were selected for semi-quantitative RT-RCR. The results of differences in gene transcription by RT-PCR correlated well with the SSH data. Significant increases in Fs29 (novel ESTs)、Fs59 (phosphoenolpyruvate carboxykinase, PEPCK) and Fs70 (novel ESTs) transcription were observed in the livers of silver carp at 1 h after exposed to MC-LR. There were significant decreases in the transcription of Rs15 (novel ESTs) and Rs161 (protein disulfide isomerase family A, member 3, PDIA3) at 1 h; however, only a slight decrease in the transcription of Rs2 (novel ESTs) was observed at 1 h. This result has proved that we successfully construct the subtracted library.4. Cloning of PEPCK and PDIA3 genes of silver carp and bioinformatics analysis Rapid amplification of cDNA ends (RACE) were used to extend the full length of two ESTs (PEPCK and PDIA3) sequence.PEPCK of Silver carp possessed 2605 base pair (bp) nucleotide that comprises of a 5’-UTR (un-translated region) of 101bp, a 3’-UTR of 564bp and a1911bp open reading frame (ORF) which encoded 636 amino acids. Sequence analysis showed that the nucleotides and amino acids sequence identity of silver carp PEPCK are highest (97% and 99% respectively) with Erythroculter ilishaeformis, secondly with Danio rerio (more than 90%), and lowest with Platichthys stellatus (77% and 84% respectively), implying PEPCK is highly conserved during the long course of evolution. Silver carp PEPCK has combined with oxalyl ethyl specific domain, as well as three chain phosphate of GTP-binding kinase 1 and kinase 2 motifs.PDIA3 of Silver carp possessed 2102bp nucleotide that comprises of a 5’-UTR of 206bp, a 3’-UTR of 381bp and a1488bp ORF which encoded 495 amino acids. Sequence analysis showed that the nucleotides and amino acids sequence identity of silver carp PDIA3 are higher (86% and 92% respectively) with Danio rerio, lower with Ictalurus punctatus (more than 80%), implying PDIA3 is highly conserved during the long course of evolution. PDIA3 includes 4 thioredoxin regions may plays a role in the formation of disulfide bonds at the time of de novo synthesis of GSH and regeneration protein.