Study On The Cloning And Expression Of Fatty Acid Binding Protein Of Ovate Pomfret Larvae And Juvenile Fish

Fatty acid binding protein(FABPs)is a set of multiple source of small molecule protein in cells with the molecular weight of 12-16 kda,widely distributed in mammalian,intestine,liver,heart,brain,skeletal muscle and other tissues.The main functions of fatty acid binding protein include fatty acid transport,cell proliferation and angiogenesis.In fish,we have found several types of fatty acid-binding proteins such as I-FABP,H-FABP,L-FABP,K-FABP,different types of fatty acid-binding protein sequence homology is relatively large.The gene for fatty acid binding proteins(FABP)in golden pompano Trachinotus ovatus larvae was cloned and analyzed from hatch to 18 days-post hatch(DPH).(1)The I-FABP gene(Gen Bank accession: MF034871)of golden pompano was composed of 815 bp with an open reading frame of 399 bp,encoded one amino acid with a molecular weight of 15.24 k Da.The predicted amino acid sequence of I-FABP genes from golden pompano showed high similarity and identity with Japanese sea bass Lateolabrax japonicus(97% and 87.9%,AOW69620.1).The highest tissue expression of I-FABP genes was found in the intestine,followed by the eye on 18 DPH.During the ontogenetic development,the expression of I-FABP genes remained a low level in the first five days,and reached the highest level on 18 DPH.The expression of I-FABP genes was not significantly affected by environmental temperature on 12 DPH,but was significantly affected by the temperature on 18 DPH.Nutrition enhancement with algae containing high fatty acids significantly affected the expression of I-FABP genes.The highest expression was observed in the non-enriched treatment,but the lowest expression was in the Nannochloropsis feeding treatment.Results of the present study indicate that the expression of the I-FABP gene varies with environmental temperature and nutritional conditions during the ontogenetic development of golden pompano larvae.The expression of I-FABP genes may be potentially used as an indicator for assessing nutrient supply and functional development of the digestive system in fish larvae.(2)The full length of L-FABP c DNA from golden pompano(Gen Bank accession No.MF034872)was 604 bp,including a 5'-untranslated region(UTR)of 154 bp,a 3'-UTR of 69 bp and an open reading frame(ORF)of 281 bp encoding a polypeptide of 126 amino acids with a predicted molecular weight of 14.06 k Da and a theoretical isoelectric point of 8.73 The predicted amino acid sequence of L-FABP gene of golden pompano showed high similarity and identity with Epinephelus coioides(97.6% and 95.2%,ADG29164.1).The highest tissue expression of L-FABP gene was observed in fish liver on 18 DPH.Along with the ontogeny of fish larvae,the expression of L-FABP gene was low at hatching,and quickly increased with the increase of fish age from 0 DPH to 4 DPH,and reached the highest level on 12 DPH.The highest expression of L-FABP gene was observed in fish cultured at 29 oC on both 12 DPH and 18 DPH(P< 0.05).Nutrition enhancement significantly affected the expression of L-FABP gene,and the highest expression was observed in the Algamac 3080 treatment,and lowest expression was observed in the Nannochloropsis treatment.This study provides useful information on the functional mechanism of L-FABP gene in the ontogenetic development of golden pompano.(3)In this study,we examined the influences of nutrition and temperature in regulating the heart-type FABP(H-FABP)gene expression in golden pompano to understand importance of the protein in fish larval development.Firstly,H-FABPs expression during ontogeny of larvae in the first 18 days of their life was explored,and then the response of H-FABPs to water temperature(23,26,and 29�C)on 12-days post hatching(DPH)and 18 DPH were compared.On 18 DPH,the response of H-FABPs to the manipulation of nutrients was evaluated.The expression of HFABPs was higher during the endogenous larval stage,and was significantly affected by water temperature.On 12 DPH larvae expressed significantly more H-FABP at a temperature of 23 and 26�C and on the contrary,on 18 DPH expressed more H-FABP at 29�C.These results may be attributed to the improved food ingestion and digestive function of larvae after 15 DPH.The expression of H-FABP in fish larvae on 18 DPH was sensitive to nutrient manipulation.The expression level assessed was the highest when Algamac 3080 enriched Artemia nauplii was used and the lowest when fed with Nannochloropsis sp.enriched Artemia nauplii.The correlation analysis between the expression of H-FABP and fatty acids detected that only ratios of fatty acids and not single fatty acid relative quantity had a significant effect on expression of H-FA BP at the early stage of golden pompano larvae.It is known that H-FABP has a major influence on transport of lipid and fatty acid storage.This study point out that changes in environment conditions,water temperature and type of feeding,can certainly influence H-FABP expression and therefore could impact golden pompano growth rate and survival in early life.