Cloning And Expression Of Fatty Acid Synthase Gene And Comparison On Growth,Muscle Quality Of Polyodon Spathula And Aristichthys Nobilis

Based on two nutritional niche similar fishes in fresh water(Polyodon spathula and Aristichthys nobilis), this paper compared their growth, feeding rate, digestibility, and muscle quality in their postlarva, juvenile and subadult growth periods. Furthermore, it studied the clone of FAS gene of Polyodon spathula and Aristichthys nobilis and expression of FAS gene mRNA in different tissue and muscles of three growth periods, drawing the following conclusions:1. Growth comparison between Polyodon spathula and Aristichthys nobilisIn postlarva fish, juvenile fish and subadult fish, Polyodon spathula achieves higher net weight increase, daily weight increase, weight increase rate and specific growth rate than those of Aristichthys nobilis (P<0.05).2. Comparison on feeding rate and digestibility between Polyodon spathula and Aristichthys nobilisWithin the postlarva fish and juvenile fish periods, the feeding rate, overall apparent digestibility, apparent digestibility of crude protein and apparent digestibility of crude fat of Aristichthys nobilis are lower than that of Polyodon spathula respectively, while the feed coefficient and apparent digestibility of crude ash of Aristichthys nobilis are higher than those of Polyodon spathula respectively.3. Comparison on proximate compositions in muscles between Polyodon spathula and Aristichthys nobilisIn postlarva fish, juvenile fish and subadult fish periods, the muscle moisture, crude protein and crude ash content of Polyodon spathula are all lower than those of Aristichthys nobilis respectively, but the crude protein in the muscle of Polyodon spathula is significantly higher than that of Aristichthys nobilis respectively (P<0.01).4. Comparison on amino acid composition in muscle between Polyodon spathula and Aristichthys nobilisFor all three periods of postlarva fish, juvenile fish and subadult fish, there are17kinds of amino acids are detected in the muscle of Polyodon spathula and Aristichthys nobilis (Trp is undetected due to acid hydrolysis), among which Glu has the highest content and Cys reveals the lowest content. The content of all amino acids in the muscles of postlarva Aristichthys nobilis are higher than that of postlarva Polyodon spathula with extremely significant difference in His (P<0.01), significant difference in Asp, Glu, Val, Lys and Arg (P<0.05), and indistinctive difference in others (P>0.05). The content of all amino acids (except for Phe and His) in the muscles of juvenile Aristichthys nobilis are higher than those of juvenile Polyodon spathula with obvious difference in Asp, Leu and Lys(P<0.05), and indistinctive difference in others (P>0.05). Except that the content of Cys in subadult Aristichthys nobilis is significantly lower than that of subadult Polyodon spathula, all other amino acids in subadult Aristichthys nobilis are higher than that of subadult Polyodon spathula with extremely significant difference in Asp, Glu and Ala (P<0.01), significant difference in Leu and Lys (P<0.05), and indistinctive difference in others (P>0.05).For all three periods of postlarva fish, juvenile fish and subadult fish, Aristichthys nobilis has higher TAA, TEAA, TNEAA, TDAA and WDAA/WTAA in muscles than those of Polyodon spathula respectively.5. Comparison on fatty acid composition in muscle between Polyodon spathula and Aristichthys nobilisFifteen kinds of fatty acids are detected in both Aristichthys nobilis and Polyodon spathula in their postlarva and juvenile periods, among which five are SFA, four are MUFA, and six are PUFA. There are totally16fatty acids detected in both Aristichthys nobilis and Polyodon spathula in their subadult period, including six SFA, for MUFA, and six PUFA.In the postlarva, juvenile and subadult periods of both Aristichthys nobilis and Polyodon spathula, their main SFA are C16:0and C18:0, MUFA are C16:1and C18:1, and PUFA are C20:5n-3(EPA) and C22:6n-3(DHA).Except that C20:1in the muscle of postlarva Aristichthys nobilis is higher than that of postlarva Polyodon spathula (P>0.05) and C22:6n-3(DHA) in the muscle of postlarva Aristichthys nobilis is significantly higher than that of postlarva Polyodon spathula(P<0.01), the rest13fatty acids content are lower than those of postlarva Polyodon spathula with indistinctive difference in C20:0(P>0.05), obvious difference in C20:2(P<0.05) and significant difference in the rest11fatty acids (P<0.01). Based on the comparison between juvenile Aristichthys nobilis and juvenile Polyodon spathula, the former one has smaller content in14fatty acids than the later one except for C20:1which is higher than that in the muscle of juvenile Polyodon spathula. Among them, C20:2 displays indistinctive difference (P>0.05), while the rest13fatty acids display significantly difference (P<0.01). In the subadult period, all fatty acid contents in the muscle of Aristichthys nobilis are lower than that of Polyodon spathula with indistinctive difference in C17:1and C20:2(P>0.05), obvious difference in C17:0(P<0.05), and significant difference in the rest13fatty acids(P<0.01).In all postlarva, juvenile and subadult periods,∑SFA、∑MUFA、∑PUFA、∑UFA、∑SFA/∑UFA、∑n-6PUFA、∑n-3PUFA、EPA、EPA+DHA、EFA and HEFA in the muscle fatty acids of Aristichthys nobilis are lower than those of Polyodon spathula respectively.6. Comparison on mineral elements in muscle between Polyodon spathula and Aristichthys nobilisIn all postlarva, juvenile and subadult Polyodon spathula and Aristichthys nobilis, there are totally10mineral elements detected from their muscles which are K, Na, Ca, Mg, Fe, Mn, Cu, Zn, Cr and Co.The contents of K, Ca, Mg, Cu, Cr and Co in the muscle of postlarva Aristichthys nobilis are higher than those of postlarva Polyodon spathula with significant difference in K, Ca and Mg (P<0.01), obvious difference in Cr and Co (P<0.05) and indistinctive difference in Cu (P>0.05). The contents of Na, Fe, Mn and Zn in the muscle of postlarva Aristichthys nobilis are lower than those of postlarva Polyodon spathula with significant difference in Na, Fe and Mn (P<0.01), and indistinctive difference in Zn (P>0.05). The contents of K, Na, Ca, Mg, Fe, Mn, Zn and Co in the muscle of juvenile of Aristichthys nobilis are higher than that of juvenile Polyodon spathula with significant difference in Na, Ca, Mg, Fe, Zn and Co (P<0.01), obvious difference in Mn (P<0.05) and indistinctive difference in K (P>0.05). The contents of Cu and Cr in the muscle of juvenile Aristichthys nobilis are lower than those of juvenile Polyodon spathula with significant difference in Cu (P<0.05) and indistinctive difference in Cr (P>0.05). The contents of Ca, Mg, Mn and Co in the muscle of subadult Aristichthys nobilis are higher than those of subadult Polyodon spathula with indistinctive difference in Ca and Mg (P>0.05), significant difference in Mn and Co (P<0.01). The contents of K, Na, Fe, Cu, Zn and Cr in the muscle of subadult Aristichthys nobilis are lower than those of subadult Polyodon spathula with indistinctive differences (P>0.05).7. FAS gene clone of Polyodon spathula and Aristichthys nobilis and analysisHomologous cloning and RACE technology are applied to clone the cDNA full-length sequence of FAS gene of Polyodon spathula and Aristichthys nobilis. The full length of XunFAS cDNA is6651bp,270bp of5’end URT and6018bp (271-6288bp) of ORF for coding a protein composed by2005amino acid residues, and363bp of3’URT, including a polyadenylation signal AATAAA that locates on the54bp of the upstream of Poly (A) tail. It is predicted that the molecular weight of XUNFAS protein is219.11kDa, and the theoretical isoelectric point (PI) is6.19. The formula of the protein is C9682H15428N2712O2914S83with unsteady nature in solutions. The whole peptide chain shows no obvious hydrophilcity and there’s no transmembrane structure and signal peptide on N end of XunFAS protein. The full length of YONGFAS cDNA is8122bp,218bp of5’end URT and7545bp (219-7763bp) of ORF for coding a protein composed by2514amino acid residues, and359bp of3’URT, including a polyadenylation signal AATAAA that locates on the255bp of the upstream of Poly (A) tail. It is predicted that the molecular weight of YONGFAS protein is274.68kDa, and the theoretical isoelectric point (PI) is5.97. The formula of the protein is C12119H19261N3393O3668S109with unsteady nature in solutions. The whole peptide chain shows no obvious hydrophilcity and there’s no transmembrane structure and signal peptide on N end of YONGFAS protein.8. Difference expressions of FAS gene mRNA in tissues of Polyodon spathula and Aristichthys nobilisReal-time PCR (qRT-PCR) technology is applied to detect the expression of FAS gene mRNA in various tissues of Polyodon spathula and Aristichthys nobilis. In Polyodon spathula, it gets the highest expression of liver FAS gene mRNA in liver, muscles, intestinal tract, fat on enterocoelia, heart, branchia, pterygiophore, eyes, stomach and lips, followed by enterocoelia fat FAS gene mRNA (about0.33times that of liver, P<0.05), and then followed by stomach FAS gene mRNA of the least expression (about0.03times that of liver, P<0.05). The expressions of FAS gene mRNA in muscles, intestinal tract, heart, branchia, pterygiophore, eyes and lips are0.21,0.04,0.05,0.08,0.09,0.07and0.04times that of liver respectively (P<0.05). And in Aristichthys nobilis, it gets the highest expression of pharynx organs FAS gene mRNA in liver, muscles, intestinal tract, pharynx organs, heart, branchia, pterygiophore and eyes, followed by intestinal tract tissue (about0.84times that of pharynx organs (P<0.05), and then followed by branchia FAS gene mRNA of the least expression (about0.06times that of pharynx organs, P<0.05). The expressions of FAS gene mRNA in liver, muscles, heart, pterygiophore and eyes are0.503,0.50,0.22,0.36and0.13times that of pharynx organs respectively (P<0.05).9. Expressions of FAS gene mRNA in muscles of Polyodon spathula and Aristichthys nobilis in different growth periodsReal-time PCR (qRT-PCR) technology is applied to detect the expression of FAS gene mRNA in muscle tissues of Polyodon spathula and Aristichthys nobilis in different growth periods. As far as the Polyodon spathula is concerned,21g Polyodon spathula possesses the highest expression of FAS gene mRNA in its muscles, followed by615g Polyodon spathula (about0.83times that of21g Polyodon spathula, P<0.05), and then followed by163g Polyodon spathula of the least expression (about0.13times that of21g Polyodon spathula, P<0.05). And with respect to Aristichthys nobilis,530g Aristichthys nobilis owns the highest expression of FAS gene mRNA in its muscles, followed by18g Aristichthys nobilis (about0.26times that of530g Aristichthys nobilis, P<0.05), and then followed by122g Aristichthys nobilis (about0.10times that of530g Aristichthys nobilis, P<0.05).