Studies On The Cloning And Expression Of Sterol Regulatory Element Binding Protein-1 Gene In Siganus Canaliculatus

Sterol regulatory element binding proteins (SREBPs) belong to the transcription factor family of basic helix-loop-helix leucine-zipper (bHLH-zip). They are the key transcription factor in eukaryotic cell which regulates lipid metabolism. The SREBP family has three isoforms: SREBP-1a, SREBP-1c and SREBP-2. SREBP-2 is derived from SREBP-2 gene, which preferentially mediates cholesterol biosynthesis; SREBP-1a and -1c produced from a single gene SREBP-1, and SREBP-1a regulates all SREBP-responsive genes, SREBP-1c is responsible for the regulation of fatty acid biosynthesis.In fish nutrition, it is generally thought that freshwater teleost can convert 18C linoleic andα-linolenic acids into 20-22C HUFA through desaturation and elongation of fatty acids. On the contrary, marine fish are generally presumed to have a low capability or inability to bioconvert linoleic andα-linolenic acids into HUFA. Nevertheless, our previous study proved for the first time in marine teleost that Siganus canaliculatus had the ability to convert linoleic andα-linolenic acids into HUFA, and such ability was higher in low salinity (10ppt) than that in high salinity (32ppt); furthermore, our recent study also cloned three genes encoding the key enzymes (fad2, fad1, Elovl5) involving in HUFA biosythesis of S. Canalicultatus, meanwhile demonstrated that the linoleic andα-linolenic acids in diet have an enhancing effect on the gene expression of these enzymes. These indicate that the HUFA biosynthetic capability in S. canalicultatus possesses the features of both marine and freshwater fish. Therefore, S. Canalicultatus is an ideal model fish for studying the regulatory mechanisms of HUFA biosynthesis in fish.In this study, in order to study the molecular regulatory mechanisms of HUFA biosynthesis in S. canaliculatus, one gene encoding the key transcription factor SREBP-1 involving in regulation of fatty acids synthesis was cloned in S. canaliculatus. Its tissue-specific distribution and the effect of environmental salinity on its expression were also investigated. The results were shown as follows:1. The full length cDNA sequence of S. canaliculatus SREBP-1 which obtained using RT-PCR,Tail-PCR and RACE-PCR, is 3913bp in length (Genebank accession no. JF502069) and encoding a polypeptide of 1170 amino acids. It contains a 3513bp open reading frame, 200bp of 5'-UTR, 200bp of 3'-UTR (excluding polyA tail). The deduced protein sequence of S. canaliculatus SREBP-1 displayed a tapical structure of SREBP. It contains a bHLH-zip domain. The deduced protein sequence of S. canaliculatus SREBP-1 shared 78% sequence identity to Salmo salar, 75% sequence identity to Danio rerio, 54%-57% to other animal species (Gallus gallus and mammal such as Mus musculus, Sus scrofa, Homo sapiens).2. The tissue-specific distribution of SREBP-1 in S. canaliculatus was measured by RT-PCR and real-timePCR. A highest mRNA expression level was observed in brain, and the second in eye and intestine, and a relative low mRNA expression level in heart, liver, gill, spleen and muscle. These suggested that brain, eye and intestine may be the main places of SREBP-1 transcription in S. canaliculatus.3. The mRNA levels of SREBP-1 in tissues including liver, intestine, brain, eye, muscle and spleen from S. canaliculatus cultured in in different salinities were detected by real-time PCR. The result shown that the mRNA expression level of SREBP-1 in liver and spleen of high salinity (32ppt) groups is higher than that of low salinity (10ppt) groups, the formers are 1.26-fold and 1.24-fold of the latters, respectively; whereas its expression level in intestine, brain and eyes in 10ppt is 2-fold, 1.5-fold and 3.4-fold of that in 32ppt, respectively; otherwise, its expression level in muscle in 32ppt is similar to that in 10ppt. The results indicate that the effect of environmental salinity on the mRNA level of S. canaliculatus SREBP-1 may change with different tissues, and have an relatively great influence on tissues such as brain, intestine and eyes where the biosynthesis of HUFA is active.This is for the first time to obtain SREBP-1 gene in a marine teleost, and also the first time to clarify the relationship between the expression level of SREBP-1 and environmental salinity in fish. The results will not only be of important theoretic academic significance for enriching fish nutrition, especially molecular nutriology, but also be helpful for clarifying the molecular mechanisms of HUFA biosynthesis of S. Canaliculatus and in fish as well.