Study On Cloning And Expression Of SREBP Gene In Takifugu Rubripes

BackgroundAdipose tissue is the largest energy storage place for animals, plays a key role in energy homeostasis and adipose tissue development imbalances often lead to many health problems. At present, the research on adipose tissue development and regulation are of concern in human medicine and agricultural sciences. Regulation of fish meat and fat deposition is an important content in the study of fishery science. The main parts of fish fat deposition in liver, skeletal muscle and adipose tissue. Where fat is deposited may bedetermined by fatty tissue specific metabolic pathways, the pathways at the molecular levelby enzymes and gene involved in the regulation of the key. Sterol regulatory elementbinding protein-1(SREBP-1) is a keytranscription factor regulating fat metabolism in eukaryotic cells.ObjectiveIn the present study, we cloned a cDNA encoding SREBP from torafugu Takifugu rubripes and analyzed its tissue distribution and recombinant protein expression.Methods1. The ORF of Takifugu rubripes SREBP gene was amplified from total RNA of torafugu liver tissues by reverse transcription-polymerase chain reaction (RT-PCR).2. Torafugu SREBP-1 transcripts were ubiquitously expressed in the different tissues by real time RT-PCR.3. Construction of prokaryotic expression vector of PET-32/SREBP-1 in Escherichia coli Rosetta (DE3) was expressed after induced by IPTG for expression of fusion protein. The protein was identified by Western blot.Results1. The sequence analysis and tissue distribution of SREBP-1 genes of Takifugu rubripesThe complete open reading frame(ORF) of Takifugu rubripes SREBP-1 which obtained using RT-PCR, is 3330 bp in length and encoding a polypeptide of 1109 amino acids, the molecular weight is 120 ku and the isoelectric point is 8.37.The multiple sequence alignment showed that coding amino acid sequences of the obtained torafugu SREBP-1 gene had 81%,73%,72%,55%,55%,54%,54% homology with the corresponding Siganus canaliculatu, Salmo salar, Danio rerio, Homo sapien, Bos taurus, Gallus gallus, respectively. Phylogenetic tree analysis showed that torafugu SREBP-1 is closely related to Siganus canaliculatu SREBP-1 than to those from other vertebrate species. Tissue expression pattern analysis showed that, SREBP-1 in the brain of Takifugu rubripes expression level was the highest, followed by the eye, adipose tissue, spleen, gill, intestine, heart and liver, the lowest level in muscle.2. The identification of the torafugu pET-32a(+)/SREBP-1Primer design a package of Takifugu rubripes SREBP-1 gene ORF region with double enzyme cutting sites, PCR and cloned into pET-32a (+) prokaryotic expression vector, double digestion and sequencing confirmed the success of double identification, the recombinant plasmid pET-32a(+)/SREBP-1.3. The expression of the torafugu SREBP-1The recombinant vector pET-32a(+)/SREBP-1 was transformed into E coli Rosetta (DE3) using 1.0 mM IPTG for induction. The protein size was 141 ku as estimated by SDS-PAGE.4. The purification and identification of the pET-32a(+)/SREBP-1 of torafuguThe obtained supernatant was purified by 6×His Ni-NTA affinity chromatography. SDS-PAGE and Western blot showed that the molecular weight of the target protein is 141 ku, that purified fusion protein was torafugu SREBP-1.ConclusionsWe cloned the full-length cDNA encoding SREBP from torafugu liver using RT-PCR, and analyzed its tissue distribution.