匙吻鲟和鳙的生长、肌肉品质比较及FAS基因克隆与表达
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摘要
本文以营养生态位相似的匙吻鲟(Polyodon spathula)和鳙(Aristichthys nobilis)为研究对象,从稚鱼、幼鱼及亚成鱼三个生长阶段,开展了匙吻鲟和鳙生长、摄食率、消化率及肌肉品质的比较,以及匙吻鲟和鳙脂肪酸合成酶(FAS)基因的克隆和FAS基因mRNA在两种鱼不同组织、三个生长阶段肌肉中的表达等研究工作。结果如下：
1.匙吻鲟和鳙生长的比较
在稚鱼、幼鱼及亚成鱼生长阶段,匙吻鲟净增重、日增重、增重率和特定生长率均显著高于鳙(P<0.05)。
2.匙吻鲟和鳙摄食率和消化率的比较
鳙稚鱼和幼鱼摄食率、总表观消化率、粗蛋白质表观消化率和粗脂肪表观消化率均分别小于匙吻鲟稚鱼和幼鱼,鳙稚鱼和幼鱼饵料系数和粗灰分表观消化率均分别高于匙吻鲟稚鱼和幼鱼。
3.匙吻鲟和鳙肌肉常规营养成分的比较
匙吻鲟稚鱼、幼鱼及亚成鱼肌肉水分、粗蛋白质和粗灰分含量均分别低于鳙稚鱼、幼鱼及亚成鱼,但肌肉粗脂肪含量均分别极显著高于鳙稚鱼、幼鱼及亚成鱼(P<0.01)。
4.匙吻鲟和鳙肌肉氨基酸组成的比较
匙吻鲟和鳙稚鱼、幼鱼及亚成鱼肌肉均检测出17种氨基酸(Trp由于酸水解未检出)。其中Glu均为最高,Cys均为最低。
鳙稚鱼肌肉的各种氨基酸含量均高于匙吻鲟稚鱼,其中His差异极显著(P<0.01), Asp、Glu、Val、Lys和Arg差异显著(P<0.05),其他氨基酸差异均不显著(P>0.05)；鳙幼鱼肌肉的各种氨基酸含量,除Phe和His外,其余氨基酸含量均高于匙吻鲟幼鱼,其中Asp、Leu和Lys差异显著(P<0.05),其他氨基酸差异均不显著(P>0.05)；鳙亚成鱼肌肉的各种氨基酸含量,除Cys含量显著小于匙吻鲟亚成鱼外(P<0.05),其余氨基酸含量均高于匙吻鲟亚成鱼,其中Asp、Glu和Ala差异极显著(P0.01),Leu和Lys差异显著(P<0.05),其他氨基酸均差异均不显著(P>0.05)。
鳙稚鱼、幼鱼及亚成鱼肌肉总氨基酸(TAA)、总必需氨基酸(TEAA)、总非必需氨基酸(TNEAA)、总致鲜氨基酸(TDAA)和WDAA/WTAA均分别高于匙吻鲟稚鱼、幼鱼及亚成鱼。
5.匙吻鲟和鳙肌肉脂肪酸组成的比较
匙吻鲟和鳙稚鱼、幼鱼肌肉中均发现15种脂肪酸,其中包括5种饱和脂肪酸(SFA),4种单不饱和脂肪酸(MUFA),6种多不饱和脂肪酸(PUFA)。匙吻鲟和鳙亚成鱼肌肉中发现16种脂肪酸,其中包括6种饱和脂肪酸(SFA),4种单不饱和脂肪酸(MUFA),6种多不饱和脂肪酸(PUFA)。
匙吻鲟和鳙稚鱼、幼鱼及亚成鱼中,主要饱和脂肪酸(SFA)均为C16：0和C18：0,单不饱和脂肪酸(MUFA)均为C16：1和C18：1,多不饱和脂肪酸(PUFA)均为C20:5n-3(EPA)和C22:6n-3(DHA)。
鳙稚鱼肌肉除C20：1高于匙吻鲟稚鱼(P>0.05)和C22:6n-3(DHA)极显著高于匙吻鲟稚鱼外(P<0.01),其余13种脂肪酸含量均小于匙吻鲟稚鱼,其中C20：0差异不显著(P>0.05),C20：2差异显著(P<0.05),其余11种脂肪酸均差异极显著(P<0.01)；鳙幼鱼肌肉除C20：1高于匙吻鲟幼鱼(P>0.05),其余14种脂肪酸含量均小于匙吻鲟幼鱼,其中C20：2差异不显著(P>0.05),其余13种脂肪酸均差异极显著(P<0.01)；鳙亚成鱼肌肉脂肪酸含量均小于匙吻鲟亚成鱼,其中C17：1和C20：2差异不显著(P>0.05),C17：0差异显著(P<0.05),其余13种脂肪酸均差异极显著(P<0.01)。
鳙稚鱼、幼鱼及亚成鱼肌肉脂肪酸中∑SFA、∑MUFA、∑PUFA、∑SFA/∑UFA、∑n-6PUFA、∑n-3PUFA、EPA、EPA+DHA、EFA和HEFA均分别低于匙吻鲟稚鱼、幼鱼及亚成鱼。
6.匙吻鲟和鳙肌肉矿物元素组成的比较
匙吻鲟和鳙稚鱼、幼鱼及亚成鱼肌肉中均检出K、Na、Ca、Mg、Fe、Mn、Cu、 Zn、Cr和Co共10种矿物元素。
鳙稚鱼肌肉中K、Ca、Mg、Cu、Cr和Co的含量高于匙吻鲟稚鱼,其中K、Ca和Mg差异极显著(P<0.01),Cr和Co差异显著(P<0.05),Cu差异不显著(P>0.05)；鳙稚鱼肌肉中Na、Fe、Mn和Zn的含量低于匙吻鲟稚鱼,其中Na、Fe和Mn差异极显著(P<0.01),Zn差异不显著(P>0.05)；鳙幼鱼肌肉中K、Na、Ca、Mg、Fe、Mn、Zn和Co的含量高于匙吻鲟幼鱼,其中Na、Ca、Mg、Fe、Zn和Co差异极显著(P<0.01),Mn差异显著(P<0.05),K差异不显著(P>0.05)；鳙幼鱼肌肉中的Cu和Cr含量低于匙吻鲟幼鱼,其中Cu差异显著(P<0.05),Cr差异不显著(P>0.05)；鳙亚成鱼肌肉中Ca、Mg、Mn和Co的含量高于匙吻鲟亚成鱼,其中Ca和Mg差异不显著(P>0.05),Mn和Co差异极显著(P<0.01)；鳙亚成鱼肌肉中的K、Na、Fe、Cu、Zn和Cr含量低于匙吻鲟亚成鱼,但差异均不显著(P0.05)。
7.匙吻鲟和鳙FAS基因的克隆与分析
采用同源克隆和RACE技术克隆了匙吻鲟和鳙FAS基因cDNA全长序列。XUNFAS cDNA全长为6651bp,5’端非翻译区(URT)270bp,开放阅读框6018bp(271-6288bp),编码一个2005个氨基酸残基组成的蛋白质,3'URT363bp且包含一个聚腺苷酸化信号AATAAA,这一信号位于Poly(A)尾巴上游54bp处；预测XUNFAS蛋白分子量为219.11kDa,理论等电点(PI)为6.19,该蛋白分子式为C9682H15428N2712O2914S83,该蛋白在溶液中性质不稳定,整条肽链没有明显的亲水性,XUNFAS蛋白N端无跨膜结构和信号肽。YONGFAS cDNA全长为8122bp,5’端非翻译区(URT)218bp,开放阅读框7545bp(219-7763bp),编码一个2514个氨基酸残基组成的蛋白质,3'URT359bp且包含一个聚腺苷酸化信号ATTAAA,这一信号位于Poly(A)尾巴上游255bp处,预测YONGFAS蛋白分子量为274.68kDa,理论等电点(PI)为5.97,该蛋白分子式为C12119H19261N339303668S109,该蛋白在溶液中性质不稳定,整条肽链没有明显的亲水性,YONGFAS蛋白N端无跨膜结构和信号肽。
8.匙吻鲟和鳙FAS基因mRNA组织差异性
通过荧光定量PCR技术分别检测了FAS基因mRNA在匙吻鲟和鳙各组织的表达。在匙吻鲟肝脏、肌肉、肠道、腹腔脂肪、心脏、鳃、鳍条、眼睛、胃和吻中,肝脏FAS基因mRNA表达量最高,其次是腹腔脂肪(约为肝脏的0.33倍,P<0.05),而胃FAS基因mRNA表达量最低(约为肝脏的0.03倍,P<0.05),肌肉、肠道、心脏、鳃、鳍条、眼睛和吻FAS基因mRNA表达量分别为肝脏的0.21、0.04、0.05、0.08、0.09、0.07和0.04倍(P<0.05)。在鳙肝脏、肌肉、肠道、咽上器官、心脏、鳃、鳍条和眼睛中,咽上器官FAS基因mRNA表达量最高,其次是肠道组织(约为咽上器官的0.84倍,P<0.05),而鳃FAS基因mRNA表达量最低(约为咽上器官的0.06倍,P<0.05),肝脏、肌肉、心脏、鳍条、眼睛FAS基因mRNA表达量分别为咽上器官的0.503、0.50、0.22、0.36和0.13倍(P<0.05)。
9.匙吻鲟和鳙肌肉FAS基因mRNA在不同生长阶段表达
通过荧光定量PCR技术分别检测了不同生长阶段匙吻鲟和鳙肌肉组织FAS基因mRNA的表达情况。21g匙吻鲟FAS基因mRNA表达量最高,其次为615g匙吻鲟(约为21g匙吻鲟肌肉组织的0.83倍,P<0.05),163g匙吻鲟FAS基因mRNA表达量最低(约为21g匙吻鲟肌肉组织的0.13倍,P<0.05)。530g鳙FAS基因mRNA表达量最高,其次为18g鳙(约为530g鳙肌肉组织的0.26倍,P<0.05),122g鳙FAS基因mRNA表达量最低(约为530g鳙肌肉组织的0.10倍,P<0.05)。
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 nobilis
In 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 nobilis
Within 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 nobilis
In 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 nobilis
For 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 nobilis
Fifteen 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 nobilis
In 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 analysis
Homologous 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 nobilis
Real-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 periods
Real-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).
1.匙吻鲟和鳙生长的比较
在稚鱼、幼鱼及亚成鱼生长阶段,匙吻鲟净增重、日增重、增重率和特定生长率均显著高于鳙(P<0.05)。
2.匙吻鲟和鳙摄食率和消化率的比较
鳙稚鱼和幼鱼摄食率、总表观消化率、粗蛋白质表观消化率和粗脂肪表观消化率均分别小于匙吻鲟稚鱼和幼鱼,鳙稚鱼和幼鱼饵料系数和粗灰分表观消化率均分别高于匙吻鲟稚鱼和幼鱼。
3.匙吻鲟和鳙肌肉常规营养成分的比较
匙吻鲟稚鱼、幼鱼及亚成鱼肌肉水分、粗蛋白质和粗灰分含量均分别低于鳙稚鱼、幼鱼及亚成鱼,但肌肉粗脂肪含量均分别极显著高于鳙稚鱼、幼鱼及亚成鱼(P<0.01)。
4.匙吻鲟和鳙肌肉氨基酸组成的比较
匙吻鲟和鳙稚鱼、幼鱼及亚成鱼肌肉均检测出17种氨基酸(Trp由于酸水解未检出)。其中Glu均为最高,Cys均为最低。
鳙稚鱼肌肉的各种氨基酸含量均高于匙吻鲟稚鱼,其中His差异极显著(P<0.01), Asp、Glu、Val、Lys和Arg差异显著(P<0.05),其他氨基酸差异均不显著(P>0.05)；鳙幼鱼肌肉的各种氨基酸含量,除Phe和His外,其余氨基酸含量均高于匙吻鲟幼鱼,其中Asp、Leu和Lys差异显著(P<0.05),其他氨基酸差异均不显著(P>0.05)；鳙亚成鱼肌肉的各种氨基酸含量,除Cys含量显著小于匙吻鲟亚成鱼外(P<0.05),其余氨基酸含量均高于匙吻鲟亚成鱼,其中Asp、Glu和Ala差异极显著(P0.01),Leu和Lys差异显著(P<0.05),其他氨基酸均差异均不显著(P>0.05)。
鳙稚鱼、幼鱼及亚成鱼肌肉总氨基酸(TAA)、总必需氨基酸(TEAA)、总非必需氨基酸(TNEAA)、总致鲜氨基酸(TDAA)和WDAA/WTAA均分别高于匙吻鲟稚鱼、幼鱼及亚成鱼。
5.匙吻鲟和鳙肌肉脂肪酸组成的比较
匙吻鲟和鳙稚鱼、幼鱼肌肉中均发现15种脂肪酸,其中包括5种饱和脂肪酸(SFA),4种单不饱和脂肪酸(MUFA),6种多不饱和脂肪酸(PUFA)。匙吻鲟和鳙亚成鱼肌肉中发现16种脂肪酸,其中包括6种饱和脂肪酸(SFA),4种单不饱和脂肪酸(MUFA),6种多不饱和脂肪酸(PUFA)。
匙吻鲟和鳙稚鱼、幼鱼及亚成鱼中,主要饱和脂肪酸(SFA)均为C16：0和C18：0,单不饱和脂肪酸(MUFA)均为C16：1和C18：1,多不饱和脂肪酸(PUFA)均为C20:5n-3(EPA)和C22:6n-3(DHA)。
鳙稚鱼肌肉除C20：1高于匙吻鲟稚鱼(P>0.05)和C22:6n-3(DHA)极显著高于匙吻鲟稚鱼外(P<0.01),其余13种脂肪酸含量均小于匙吻鲟稚鱼,其中C20：0差异不显著(P>0.05),C20：2差异显著(P<0.05),其余11种脂肪酸均差异极显著(P<0.01)；鳙幼鱼肌肉除C20：1高于匙吻鲟幼鱼(P>0.05),其余14种脂肪酸含量均小于匙吻鲟幼鱼,其中C20：2差异不显著(P>0.05),其余13种脂肪酸均差异极显著(P<0.01)；鳙亚成鱼肌肉脂肪酸含量均小于匙吻鲟亚成鱼,其中C17：1和C20：2差异不显著(P>0.05),C17：0差异显著(P<0.05),其余13种脂肪酸均差异极显著(P<0.01)。
鳙稚鱼、幼鱼及亚成鱼肌肉脂肪酸中∑SFA、∑MUFA、∑PUFA、∑SFA/∑UFA、∑n-6PUFA、∑n-3PUFA、EPA、EPA+DHA、EFA和HEFA均分别低于匙吻鲟稚鱼、幼鱼及亚成鱼。
6.匙吻鲟和鳙肌肉矿物元素组成的比较
匙吻鲟和鳙稚鱼、幼鱼及亚成鱼肌肉中均检出K、Na、Ca、Mg、Fe、Mn、Cu、 Zn、Cr和Co共10种矿物元素。
鳙稚鱼肌肉中K、Ca、Mg、Cu、Cr和Co的含量高于匙吻鲟稚鱼,其中K、Ca和Mg差异极显著(P<0.01),Cr和Co差异显著(P<0.05),Cu差异不显著(P>0.05)；鳙稚鱼肌肉中Na、Fe、Mn和Zn的含量低于匙吻鲟稚鱼,其中Na、Fe和Mn差异极显著(P<0.01),Zn差异不显著(P>0.05)；鳙幼鱼肌肉中K、Na、Ca、Mg、Fe、Mn、Zn和Co的含量高于匙吻鲟幼鱼,其中Na、Ca、Mg、Fe、Zn和Co差异极显著(P<0.01),Mn差异显著(P<0.05),K差异不显著(P>0.05)；鳙幼鱼肌肉中的Cu和Cr含量低于匙吻鲟幼鱼,其中Cu差异显著(P<0.05),Cr差异不显著(P>0.05)；鳙亚成鱼肌肉中Ca、Mg、Mn和Co的含量高于匙吻鲟亚成鱼,其中Ca和Mg差异不显著(P>0.05),Mn和Co差异极显著(P<0.01)；鳙亚成鱼肌肉中的K、Na、Fe、Cu、Zn和Cr含量低于匙吻鲟亚成鱼,但差异均不显著(P0.05)。
7.匙吻鲟和鳙FAS基因的克隆与分析
采用同源克隆和RACE技术克隆了匙吻鲟和鳙FAS基因cDNA全长序列。XUNFAS cDNA全长为6651bp,5’端非翻译区(URT)270bp,开放阅读框6018bp(271-6288bp),编码一个2005个氨基酸残基组成的蛋白质,3'URT363bp且包含一个聚腺苷酸化信号AATAAA,这一信号位于Poly(A)尾巴上游54bp处；预测XUNFAS蛋白分子量为219.11kDa,理论等电点(PI)为6.19,该蛋白分子式为C9682H15428N2712O2914S83,该蛋白在溶液中性质不稳定,整条肽链没有明显的亲水性,XUNFAS蛋白N端无跨膜结构和信号肽。YONGFAS cDNA全长为8122bp,5’端非翻译区(URT)218bp,开放阅读框7545bp(219-7763bp),编码一个2514个氨基酸残基组成的蛋白质,3'URT359bp且包含一个聚腺苷酸化信号ATTAAA,这一信号位于Poly(A)尾巴上游255bp处,预测YONGFAS蛋白分子量为274.68kDa,理论等电点(PI)为5.97,该蛋白分子式为C12119H19261N339303668S109,该蛋白在溶液中性质不稳定,整条肽链没有明显的亲水性,YONGFAS蛋白N端无跨膜结构和信号肽。
8.匙吻鲟和鳙FAS基因mRNA组织差异性
通过荧光定量PCR技术分别检测了FAS基因mRNA在匙吻鲟和鳙各组织的表达。在匙吻鲟肝脏、肌肉、肠道、腹腔脂肪、心脏、鳃、鳍条、眼睛、胃和吻中,肝脏FAS基因mRNA表达量最高,其次是腹腔脂肪(约为肝脏的0.33倍,P<0.05),而胃FAS基因mRNA表达量最低(约为肝脏的0.03倍,P<0.05),肌肉、肠道、心脏、鳃、鳍条、眼睛和吻FAS基因mRNA表达量分别为肝脏的0.21、0.04、0.05、0.08、0.09、0.07和0.04倍(P<0.05)。在鳙肝脏、肌肉、肠道、咽上器官、心脏、鳃、鳍条和眼睛中,咽上器官FAS基因mRNA表达量最高,其次是肠道组织(约为咽上器官的0.84倍,P<0.05),而鳃FAS基因mRNA表达量最低(约为咽上器官的0.06倍,P<0.05),肝脏、肌肉、心脏、鳍条、眼睛FAS基因mRNA表达量分别为咽上器官的0.503、0.50、0.22、0.36和0.13倍(P<0.05)。
9.匙吻鲟和鳙肌肉FAS基因mRNA在不同生长阶段表达
通过荧光定量PCR技术分别检测了不同生长阶段匙吻鲟和鳙肌肉组织FAS基因mRNA的表达情况。21g匙吻鲟FAS基因mRNA表达量最高,其次为615g匙吻鲟(约为21g匙吻鲟肌肉组织的0.83倍,P<0.05),163g匙吻鲟FAS基因mRNA表达量最低(约为21g匙吻鲟肌肉组织的0.13倍,P<0.05)。530g鳙FAS基因mRNA表达量最高,其次为18g鳙(约为530g鳙肌肉组织的0.26倍,P<0.05),122g鳙FAS基因mRNA表达量最低(约为530g鳙肌肉组织的0.10倍,P<0.05)。
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 nobilis
In 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 nobilis
Within 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 nobilis
In 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 nobilis
For 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 nobilis
Fifteen 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 nobilis
In 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 analysis
Homologous 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 nobilis
Real-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 periods
Real-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).
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