Cloning And Effects Of Dietary Lipid And Choline And Feeding Frequency And Levels On Expression Of Fatty Acid Synthase And Catalase In Liver Of Juvenile GIFT, Oreochromis Niloticus

Fatty acid synthase （FAS） is a key enzyme in the synthesis of fat, which catalyzes the acetyl coenzyme A and malonyl coenzyme A to synthetic fatty acid, and then synthetic the fat with glycerol, FAS plays an important role in the deposition of animal fat. Catalase （CAT） is a widely present in plants and animals of oxidases, which is mainly used to decompose the H2O2 of body, avoiding the oxidative damage of biological macromolecules. In the current study, we focused on GIFT as models to clone the full-longth cDNA sequences of FAS and CAT in liver by RT-PCR. By qRT-PCR method to analyze the different dietary lipid levels and choline levels, feeding frequency and feeding levels, and the relative expression levels of FAS and CAT gene in liver. The main achievements of our studies were asfollowing:1. This experiment has been cloned FAS and CAT full-length cDNA whoes fragment size were 8779 bp and 3063 bp respectively, encoding 2511 and 527 amino acid residues respectively. The FAS and CAT of GIFT amino acid sequence was aligned with others similarities were 61%-78%and 67%-92% respectively. The deduced amino acid sequence of GIFT presents highly conserved signature sequences with other species. FAS contains the ketoacyl synthase active site C161, palmitic acid thiolase activity peptide "-SFGACVAF-" and enoyl reductase activity area "-VFTTVGSMEKR-", CAT contains heme binding site "-RLFSYPDTH-" and the enzyme active site sequence "-FDRERIPERVVHAKGAG-", three amino acid residues involved in catalysis （His75, Asn148, Tyr358）, there are 12 NADPH binding site and a peroxisomal targeting signal "SKM" in C-terminal. The phylogenetic tree results show that molecular biology classification is consistent with the traditional method. Bioinformatics analysis show that, FAS and CAT proteins are free from the transmembrane domain and the signal peptide, subcellular localization is in the cytoplasm, suggesting that they are non-secreted protein. Domains forecast, FAS protein contains an N-terminal and C-terminal domain of β-ketoacyl synthases, （ketoacyl-synt） and （Ketoacyl-synt_C）, acyltransferase domain （Acyl_transf₁）, dehydratase domain （PS-DH）, alkenyl reductase domain （PKS_ER）, further containing keto reductase domain （PKS_KR）, phosphopantetheine mercaptoethylamine binding site （PP-binding） and the thioesterase domain （Thioesterase）. CAT speculated protein containing heme-catalase core domain （Catalase domain） and catalase-related immune response sequence （Catalase-rel）. Protein secondary structure analysis shows that FAS and CAT secondary structure both consist of alpha helix, extended strand and random coil with a ratio of them were 35.84%,18.36%,45.80%and 29.41%,19.17%,51.42%respectively. Conclusion:This experiment has been cloned FAS and CAT full-length cDNA, bioinformatics analysis showed that FAS and CAT gene amino acid sequences and domains have a higher conservation among different species, the encoded proteins are cytoplasmic proteins.2. In order to study the affect of different levels of dietary lipid and choline to the expression of FAS and CAT in liver of Juvenile GIFT, this test detect the expression of FAS and CAT which come from nine groups with different fat levels and choline levels by qRT-PCR, the fat level and choline level was:group 1:4%and 500 mg·Kg-1, group 2:4%and 750 mg·Kg-1, group 3:4%and 1000 mg·Kg-1, group 4:8%and 500 mg-Kg-1, group 5:8%and 750 mg·Kg-1, group 6: 8%and 1000 mg·Kg-1, group 7:12% and 500 mg-Kg-1, group 8:12%and 750 mg·Kg-1, group 9:12% and 1000 mg·Kg-1. Each group had 3 replicates with 50 fish per replicate. After feeding for 63d, The results showed that:when the level of dietary choline is 500mg·Kg-1, the expression of FAS in liver decreased gradually with increasing fat levels, while choline level is 750 mg·Kg-1, with increasing fat levels, the expression of FAS increased significantly （P<0.05）, the expression of FAS reached a peak at the fat level of 8% when choline was a higher level （1000 mg-Kg-1）; when choline level were 750 mg-Kg-1 and 1000 mg·Kg-1, with the increasing fat level, the expression of CAT in liver is increased but then decreased, group of 8% fat was significantly higher than 4% group and 12%group （P<0.05）, while the expression of CAT significantly increased （P<0.05） with the increase of the fat level in the lower levels of choline （500 mg·Kg-1）. Results suggest that increasing dietary fat level at lower levels of choline may inhibit the expression of FAS, add the right amount of choline can eliminate the inhibition, but an excess of choline level will also express inhibition to FAS at high-fat group; An appropriate increasion of dietary lipid level will increase anti-oxidant capacity of Juvenile GIFT, but excess fat level will get the opposite result, while lower levels of choline （500 mg·Kg-1） will activate a large amount of expression of CAT at high-fat group.3 In order to investigated the effects of feeding frequency and feeding levels on the expression of FAS and CAT in liver Juvenile GIFT, this test detect the expression of FAS and CAT which come from ten groups with different feeding frequency and feeding levels by qRT-PCR. The feeding frequency and feeding levels were:2·d-1 and 2%,2·d-1 and 4%,2·d-1 and 6%,2·d-1 and 8%, 2·d-1 and 10%,3·d-1 and 2%,3·d-1 and 4%,3·d-1 and 6%,3·d-1 and 8%,3·d-1 and 10%. Each group had 3 replicates with 60 fish per replicate. After feeding for 70d, The results showed that:When the feeding frequency was twice a day, the expression of FAS in liver increased but then decreased with increasing feeding levels, while increased again at the feeding level is 10%, the difference among groups 4%,6%,8%was not significant （P>0.05）, but group 6%was significantly higher than group 2%（P<0.05）, but significantly lower than the group 10%（P<0.05）; When fed three times a day, the trendency of increased but then decreased and then increased of FAS expression with elevated feeding levels was more obvious except group 2%, group 6% was significantly higher than group 4%and group 8%（P<0.05）, but significantly lower than the group 10%（P<0.05）; When fed twice a day, with the increased levels of feeding, the expression of the CAT gene in liver decline and reach the minimum at group 8% at first, and then rised; At three times a day, except group 2%, other groups the trendency of decreased but then increased was somewhat in advance, it reached the peak at group 6%. The results suggest that:fed with increased levels, the expression of FAS in liver of Juvenile GIFT was wave-like increasing tendency, and the increased feeding frequency enhances the tendency; The expression of CAT in liver with feeding levels rise up after decreased, the increasion of feeding frequency make this trendency in advance, while inhibited the expression at low feeding levels.