| The aim of this paper was to study the low temperature adaptation mechanisms of the freshwater fish at gene expression level, we selected Heilongjiang common carp from Northeast China's as the experimental material, which is the cold-tolerant species. According to the double standard-curve method, real-time fluorescence quantitative PCR was performed to analyze the relative expression levels in the brain of this fish species. In view of the brain is not only the nerve center, but also an important tissue involved in regulation of body temperature, while the variety of gene expression is usually the fastest and most sensitive in the brain, so we collect the brain as subject. According to the previous reports, we collected and download 50 EST sequences related to the cold induction of common carp, then designed their primers according to the characteristics and requirements of quantitative RT-PCR that product size is between 100 bp and 200 bp. Common carp were divided into two groups, the control group and experimental group. The experimental carps were acclimated to 6℃for 10 days, while its couterpart (23℃) were subjected to an identical handling regime. At prescribed time points, fish were sampled, then RNA was isolated and reverse transcripted. Two cDNA templates of carp were randomly picked and amplified by a set of 50 primers, Fortunately, 26 candidate genes can be amplified with only one clear bands. RT-PCR was performed on Rotor gene 3000 instrument, 26 candidate genes were quantified and normalized in the brain cDNA of common carp at 23℃and 6℃using double-standard curve method of real-time quantitative PCR. The results showed that five candidates up-regulated in the samples at 6℃(P<0.01) and quantified 2.11, 13.9, 2.52, 7.38, and 1.83 times more than in the samples at 23°C, respectively. Gene function searching indicated that the protein products of these five candidates were elongation of fatty acid elongase(ELO), Acyl-CoA desaturase(SCD), Transcription initiation factor IIB(TFIIB), Myo-inositol-1-phosphate synthase(MIPS), and Blood-brain barrier HT7 antigen (bsg) individually. Moreover, seven down-regulated candidates were also identified in the same samples at 6℃(P>0.05), and their expression levels were decreased by 21.8%, 25.9%, 16.6%, 23.7%, 15.8%, 16.3%, and 42.5%, respectively, in comparison with the samples at 23℃. These seven down-regulated candidates mainly participated in the inhibition of glycolysis, improvement of cell apoptosis, and intervention of synapse remodeling based on the results of function searching.Homologous sequences of the five candidate genes can be identified using blast approach query against the NCBI zebrafish(Danio rerio) database, as result of only four candidate genes were finally defined. Subsequently, four candidate genes were quantified and normalized in the brain cDNA of zebrafish at 23℃and 10℃using the same method as described above. The result suggested that four genes that encode the ELO, SCD, TFIIB and bsg increased quantitatively 0.76 times, 1.62 times, 1.36 times and 1.05 times, at 10℃in comparison with 23℃, however, there were not significantly different(P> 0.05). The real-time quantitative PCR results of carp and zebrafish revealed that the five candidate genes were most likely related to fish with cold tolerance, which involved in unsaturated fatty acid metabolism, nervous system regulation, transcriptional regulation and material exchange and transportation in the brain of fish species at cooling water temperatures.The five cold-induced genes identified in this study will be used as important elements for fish with cold sensitive through transgenic technology in future. SMARTer? RACE cDNA Amplification Kit was successfully used for cloning the 5' end and 3'-end of the five genes, combined with the DNAstar-SeqMan software to reconstruct the full-length cDNA with separate but overlapping 3' and 5' RACE PCR products following sequencing. These five full-length cDNA sequences were deposited to NCBI and gained the GenBank accession numbers, they were JF836160, JF836162, JF836163, JF836164 and JF836165. The length of five cDNAs were 2737bp, 2618bp, 1555bp, 2154bp and 1971bp, respectively. Using ORF finder, we identified the ORF of the full-length cDNA sequences, the length of frame were 876bp, 975bp, 951bp, 1659bp and 840 bp individually, which can be translated to291aa, 324aa, 316aa, 552aa and 279aa accordingly. We predicted the primary and secondary structures with the help of protparam, protscale, TMHMM and PHD softwares, and found functional domain using InterPro software, and built the homology models by the SWISS-MODEL server. InterPro search result showed that the five protein belong to fatty acid elongase, fatty acid desaturase, transcription initiation factor IIB, myo-inositol-1-phosphate synthase and basign protein family sequences. SWISS-MODEL workspace provide homology models for Transcription initiation factor IIB and Myo-inositol-1-phosphate synthase, so the 3D structure of fatty acid elongase, fatty acid desaturase and basign protein need more deepview. The different branch length in the phylogenetic trees constructed by the N-J metod indicated difference of evolutionary rates in the process of evolution, five trees indicates that carp own the similar amino acid contents in proteins with the Atlantic salmon(Salmo salar) of cold-water fish and grass carp(Ctenopharyngodon idellus) of warm-water fish but have a distance with zebrafish and milkfish(Chanos chanos) of tropical fish. Therefore, we suggested that these proteins were likely to be closely related to cold adaptability of fish.In addition, we reconstructed transfer vector with Tol2 transposon system for the following transgenic experiment. Oweing to the restrictions of restriction enzyme cutting sites, we just inserted the fatty acid desaturase ORF into Tol2 vector. |
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