草鱼D-loop多态性与幼苗生长性状的关联分析

为了探索草鱼(Ctenopharyngodon idella)线粒体DNA(mitochondrial DNA,mt DNA)多态性对生长性状的影响,鉴于mt DNA的母性遗传特征,本研究基于2011年繁殖用的20尾母本的D-loop序列信息,与通过亲子鉴定获得的853尾40日龄子代的体长、体质量进行关联分析。结果显示,草鱼6种D-loop单倍型对生长性状表型差异具有极显著影响(P0.01);其中,单倍型为Hap16的子代的体长最大,并显著大于单倍型为Hap4的子代的体长(P0.05);单倍型为Hap18和Hap16的子代的体质量较大,依次大于其他单倍型子代的体质量,并显著大于单倍型为Hap4的子代的体质量(P0.05)。此外,草鱼D-loop序列各变异位点基因型对生长性状的影响水平不同;其中,Site01、Site06和Site07等3个位点对体长的差异存在显著影响(P0.05),Site06和Site07等2个位点对体质量的差异存在显著影响(P0.05)。研究表明,草鱼D-loop序列变异对子代生长性状具有显著影响,推测在草鱼生长性状改良的选育进程中,可以利用mt DNA多态性信息进行辅助选择。     

投饲蚕豆对草鱼生长和肉质影响的初步研究

分别以配合饲料(对照组)、蚕豆(蚕豆组)饲喂平均体重为496.5 g的草鱼(Ctenopharyngodon idella)66d,考察蚕豆对草鱼生长和肉质的影响。结果表明:配合饲料组和蚕豆组增重率为82.76%,36.96%;饵料系数为2.17,4.45;肥满度为13.09,7.72;粗脂肪含量为0.89%,1.43%;肌肉失水率为27.14%,15.87%;肌原纤维长度为28.32μm,33.31μm;羟脯氨酸0.12mg/g,0.16mg/g。在生长方面,与配合饲料组相比,蚕豆组草鱼的增重率、肥满度极显著降低,饲料系数极显著增加(P<0.01);在肌肉品质方面,失水率极显著降低;粗脂肪含量、肌原纤维长度、羟脯氨酸含量极显著提高(P<0.01)。结果表明,投喂蚕豆对草鱼肌肉具有特殊的改变作用。     

饥饿对草鱼血清游离氨基酸的影响

将池塘捕获的体重(1.70±0.80)kg的草鱼(Ctenopharyngodon idella)饲养在室内水族箱内,分别测定了空腹(禁食1 d,26样本)、饥饿15 d(15样本)、饥饿30 d(15样本)的草鱼血清游离氨基酸含量。结果显示:草鱼血清游离氨基酸总量基本恒定,空腹、饥饿15 d和饥饿30 d的氨基酸总量无显著差异;处于饥饿状态的草鱼血清游离必需氨基酸总量极显著地高于空腹状态,非必需氨基酸总量极显著地低于空腹状态;血清赖氨酸、缬氨酸、异亮氨酸、亮氨酸和胱氨酸含量因饥饿显著升高,而组氨酸、苯丙氨酸、天冬氨酸、丝氨酸、谷氨酸和酪氨酸因饥饿显著降低;饥饿15 d和饥饿30 d各氨基酸含量均未见差异。草鱼血清游离氨基酸含量在正常摄食情况下主要受饲料蛋白质影响,处于饥饿状态下主要来源于肌体蛋白质的分解,比较正常摄食后和饥饿状态下血清游离氨基酸的组成可评价饲料蛋白质的质量。     

镁对草鱼生长、形体、肝功能和糖代谢的影响

在以酪蛋白和明胶为蛋白源的纯化日粮(镁含量76.9 mg/kg)中添加不同水平的镁(0、150、300、600、1200和2400 mg/kg),研究镁对体重为(10.68±0.14)g的草鱼(Ctenopharyngodon idella)生长、形体、肝功能和糖代谢的影响,时间为10周.结果显示:日粮中适当添加镁显著...     

草鱼不同部位肌肉营养成分、肌纤维特性以及脂肪代谢相关基因的表达

为了探讨草鱼(Ctenopharyngodon idella)不同部位肌肉品质的差异,本研究选取体质量为(1 026.10±145.55)g的草鱼,检测了背部、腹上部、腹底部和尾部肌肉的营养成分、肌纤维组织结构特性以及脂肪代谢相关基因的表达。结果表明,草鱼腹底部肌肉的粗脂肪含量显著高于背部、腹上部和尾部肌肉,而水分含量则显著降低;背部肌肉粗蛋白含量显著高于腹上部、腹底部和尾部肌肉。腹上部肌肉的肌纤维直径显著高于背部、腹底部和尾部肌肉,但其肌纤维密度则显著降低,而最高的肌纤维密度出现在腹底部肌肉;草鱼背部和尾部肌肉肌纤维密度无显著差异。草鱼腹底部肌肉的脂蛋白脂肪酶基因mRNA表达量显著高于草鱼背部、腹上部和尾部肌肉。脂肪酸合成酶和乙酰辅酶A羧化酶α的mRNA表达量在草鱼背部、腹上部、腹底部和尾部肌肉之间无显著差异。研究表明,草鱼不同部位的肌肉营养特性存在显著性差异,肌肉脂肪含量与肌纤维直径呈负相关,与脂蛋白脂肪酶基因的表达量呈正相关。     

投饲率对草鱼生长、体组成和能量收支的影响

为了建立草鱼(Ctenopharyngodon idella)的精准投喂模型管理系统,研究了不同投喂水平下草鱼的能量收支。实验在水温(27.5±2.0)℃条件下,选用初始体重(71.03±1.13)g的草鱼,共设5个不同投饲水平(饥饿、1%鱼体重(1%BW)、2%BW、4%BW和饱食),研究摄食水平对草鱼生长、鱼体组分和能量收支的影响。结果显示:随投喂水平的增加,草鱼鱼体水分减少,粗蛋白含量、粗脂肪含量和能量增加,灰分含量先减少后增加。特定生长率和热积温生长系数均随投喂水平的增加先增加再降低,在5%鱼体重时达到最大,且高于饱食投喂组。饵料转化率(FE)在投喂水平为体重2%组最高,且显著高于其他各组。干物质和能量表观消化率随投喂水平的增加而显著增加,蛋白质表观消化率在投喂水平为体重2%组显著高于其他组。生长能比例随投喂水平的增加而显著增加,而在体重2%组到饱食组间均无显著性差异;代谢能与生长能相反。在最大投喂水平下的能量收支方程为:100 C=21.72 F+4.25 U+45.85R+28.18 G或100 A=61.94R+38.06G。结果表明,5%鱼体重的投喂量为草鱼此阶段的最佳投喂水平。     

玻璃红鲤MHCⅡ类基因多态性与组织表达分析

以cDNA为模板进行PCR扩增和克隆分析,从7尾玻璃红鲤(Cyprinus carpio var.wananensis)的70个有效克隆中,获得长度为624 bp的MHC IIα/β不同编码序列25/31个,分属于7/10个不同等位基因,揭示玻璃红鲤MHC II类基因的多态性仍较丰富。其中Cyca-DXA17*01和Cyca-DAB1*1201、Cyca-DAB1*1301、Cyca-DAB3*1901、Cyca-DAB3*2001、Cyca-DAB3*2101、Cyca-DAB3*2201为新发现的7个基因。分析表明,α1/β1结构域的核苷酸/氨基酸变异位点数明显高于α2/β2结构域;α1、β1结构域抗原结合位点(PBR)的ω值(ω=dN/dS,5.209和1.703)都大于1,揭示玻璃红鲤MHC II类基因在进化过程中受到正向选择作用。半定量分析显示,玻璃红鲤MHC II类基因在肾和脾中有较强表达,中等程度表达于胃、肝和肠,而鳃、心和眼的表达较弱。     

长江草鱼多态微卫星位点的分离及后备亲鱼的遗传多样性分析

本研究采用磁珠富集法分离了10对具有多态性的草鱼(Ctenopharyngodon idellus)微卫星位点,并对140条长江野生草鱼组成的后备亲鱼群体的遗传结构进行了分析。结果显示:本试验得到的草鱼微卫星序列主要是以2个碱基为重复单位、重复次数在5～22之间的多重复单元,重复次数在10次以上的微卫星序列较易得到多态性;77个微卫星座位中,完美型、非完美型和混合型微卫星标记所占的比例分别为87.01%、2.60%和10.39%;群体遗传分析显示,10个多态性微卫星座位中,除了GC39座位外,其它座位都符合哈迪-温伯格平衡,适用于草鱼群体的遗传结构分析;每个座位检测到3～8个等位基因,平均有效等位基因数为3.9302,多态信息含量在0.4129～0.8107之间变动,平均为0.6678,除了GC78座位为中度多态外,其他9个座位均为高度多态。基因分化系数、Shannon指数、平均观测杂合度和平均期望杂合度的平均值分别为0.3457、1.4262、0.9511和0.7193,表明该群体的遗传多样性比较丰富。     

中华鳖群体间编码MHCⅠ类分子α2结构域基因的克隆及序列分析

通过主要组织相容性复合体（MHC）基因的分析,探讨了中华鳖5个群体（黄河、淮河、洞庭湖、鄱阳湖、太湖）间的遗传变异与分化。中华鳖编码MHCⅠ类分子α2结构域基因的多态性很丰富。主要表现在：（1）中华鳖编码MHCⅠ类分子α2结构域的基因序列存在插入或缺失变异,共获得7种不同长度的基因序列,其中222bp、231bp分别在42、23个体出现,为两种主要长度的基因序列;（2）共获得41种不同的核苷酸序列,这些核苷酸序列的相似度在0．386—0．995之间,表明序列间的歧异度较大;（3）在所有核苷酸序列中,共有250个有效位点,其中174个为变异位点,多态位点百分率达69．6％,但群体间存在差异：①多态位点百分率大小顺序为鄱阳湖鳖（71．02％）〉太湖鳖（58．05％）〉淮河鳖（57．69％）〉黄河鳖（55．32％）〉洞庭湖鳖（48．09％）;②核苷酸多样性指数（π）大小顺序为鄱阳湖鳖（0．4273）〉太湖鳖（0．2872）〉洞庭湖鳖（0．2840）〉黄河鳖（0．2727）〉淮河鳖（0．2463）;（4）分子方差分析（AMOVA）表明,太湖鳖与黄河鳖、淮河鳖2群体间,淮河鳖与洞庭湖鳖间有极显著的遗传分化（P〈0．001）;（5）根据核苷酸序列的平均遗传距离,用UPGMA和NJ法进行聚类分析,黄河鳖与淮河鳖、洞庭湖鳖与太湖鳖分别先聚在一起,最后再与鄱阳湖鳖聚类,显示鄱阳湖鳖与其它4群体中华鳖在MHC基因上有明显歧化。     

牙鲆MHC classⅡB基因多态性及其与鱼体抗病力关系的分析

用fmhcN1 和 fmhcC1 引物分别从 42尾感病个体和42尾抗病个体的基因组DNA中扩增MHC基因片段,扩增产物长度为268/280 bp。 在268/280 bp的核苷酸序列中,有32个(11.4%)核苷酸位点是多态的。在其编码的61个氨基酸位点中,有13个位点是多态的,其中有6个位点发生在多肽结合位点上。对核苷酸替代的类型及位点进行分析,发现非多肽结合位点的非同义碱基替代率与同义碱基替代率的比值(dN/dS) (0.523)远远小于多肽结合位点的非同义碱基替代率与同义碱基替代率的比值(dN/dS) (23.091),表明氨基酸替换集中出现在exon2多肽结合位点上。分析84个个体的411个阳性克隆的测序结果,发现有13 个不同的MHC class ⅡB等位基因,并且分别编码 13个不同的氨基酸序列。其中大部分等位基因如a, b, c, d, e,f, j, k, i, m是两个群体共有的,等位基因d 在感病群体中出现的频率（23.80％）显著高于在抗病群体中出现的频率（7.14％）。而等位基因g和h只出现在13个抗病个体中, 其频率分别为21.4% 和 9.52％;等位基因l只出现在感病群体中,其频率为 19.05%。     

Dietary available phosphorus requirement of juvenile grass carp (Ctenopharyngodon idella)

A growth trial was conducted to estimate the optimum concentration of dietary available phosphorus (P) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (5.59 ± 0.02 g) were fed diets containing graded levels (2.36, 4.27, 6.31, 8.36, 10.4 and 14.8 g kg^?1) of available P for 8 weeks. Grass carp fed with the P‐supplemented diets had significantly higher specific growth rate, weight gain, protein efficiency ratio and feed efficiency than fish fed with the basal diet. In whole‐body composition, protein content increased, while lipid content decreased with the increase in P level in diet (P < 0.05). Fish fed with the P‐supplemented diets had significantly higher whole body, vertebrae and scales mineralization (P < 0.05), but Ca/P ratios were not influenced. The blood chemistry analysis showed that dietary available P had distinct effects on P, Ca and Mg contents, as well as on the contents of triacylglycerol and total cholesterol. Broken‐line analysis indicated that 8.49 g kg^?1 dietary available P was required for maximal tissue storage and mineralization as well as optimal growth.     

草鱼C1qC基因的克隆及表达分析

为了研究C1qC基因在草鱼(Ctenopharyngodon idella)免疫过程中所起的作用,利用RT-PCR和RACE方法克隆获得了C1qC基因cDNA全长序列,经序列分析表明,所克隆的C1qC cDNA全长为916 bp,包括开放阅读框(open reading frame,ORF)735 bp,5′端非编码区(untranslated region,UTR)89 bp和3′端非编码区(UTR)92 bp。735 bp的ORF共编码244个氨基酸,相对分子量为26 162.5 U。同源性分析表明,草鱼与斑马鱼(Danio rerio)的相似度最高,达到71%。经草鱼呼肠孤病毒(grass carp reovirus,GCRV)诱导后,草鱼C1qC基因在鳃、皮肤、肌肉、肝、中肾、心脏、头肾等组织中的mRNA表达水平均显著上调。在草鱼胚胎发育的各个阶段都能检测到C1qC mRNA的表达,说明该基因可能在草鱼胚胎和鱼苗的免疫反应和早期发育中起重要作用。本研究将为今后在草鱼免疫功能方面深入研究C1qC基因提供基础资料。     

草鱼HSP70基因cDNA部分序列克隆及其组织表达差异

根据鲤热休克蛋白70(Heat shock protein,HSP70)序列(AY120894)设计并合成一对引物,以草鱼(Cteno-pharyngodon idella)肝胰脏组织总RNA为模板,RT-PCR扩增获得草鱼HSP70基因cDNA部分序列,并进行了组织表达差异性研究。结果显示:所获为序列为480 bp,获得GeneBank登陆号为FJ483832。序列测序结果显示,HSP70扩增序列与鲤、斑马鱼、鲋的同源性为:93%、91%、93%。另外,所获序列HSP70在草鱼脂肪、肌肉、肠、脑、粘液、性腺、鳔、肝胰脏、心脏、脾脏、鳃、鳍12个组织的表达存在差异,HSP70在草鱼这12个组织中均检测到表达,其中在鳍中表达最高,极显著高于其他组织(P<0.01);在鳔中表达次之,且与脑、心脏、性腺中表达差异不显著;在粘液中表达最低。     

Dietary zinc requirement of juvenile grass carp (Ctenopharyngodon idella) based on growth and mineralization

A growth trial was conducted to estimate the optimum requirement of dietary zinc (Zn) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (3.97 ± 0.05 g) were fed diets containing graded levels (13, 25, 34, 53, 89 and 135 mg kg^?1) of Zn for 8 weeks. Grass carp fed with dietary Zn levels higher than 34 mg kg^?1 significantly increased final body weight, weight gain and specific growth rate (P < 0.05). For body composition, fish fed with dietary Zn levels higher than 53 mg kg^?1 significantly decreased the moisture contents but increased the lipid contents of whole body and liver. Whole body, scales, vertebrae and liver mineralization were all affected significantly (P < 0.05) by dietary Zn levels. Zn contents in whole body, scales, vertebrae and plasma were linearly increased up to the 53 mg kg^?1 dietary Zn and then remained stable beyond this level. Grass carp fed with dietary Zn levels higher than 53 mg kg^?1 significantly increased triacyglyceride and total cholesterol contents and plasma alkaline phosphatase activity in plasma (P < 0.05). Broken‐line analysis indicated that 55.1 mg kg^?1 dietary Zn was required for maximal tissue storage and mineralization as well as optimal growth of grass carp.     

Dietary magnesium requirement and effects on growth and tissue magnesium content of juvenile grass carp (Ctenopharyngodon idella)

A growth trial was conducted to estimate the optimum concentration of dietary magnesium (Mg) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (5.56 ± 0.02 g) were fed diets containing graded levels (187, 331, 473, 637, 779 and 937 mg kg^?1) of Mg for 8 weeks. Weight gain, specific growth rate and feed efficiency were linearly increased up to 637 mg kg^?1 dietary Mg and then levelled off beyond this level. For body composition, dietary Mg levels higher than 473 mg kg^?1 significantly decreased the moisture content but increased the lipid content of whole body, muscle and liver. Dietary Mg levels higher than 473 mg kg^?1 significantly decreased the ash contents of vertebrae, scales and muscle. Mg contents in whole body, vertebrae, scales and plasma were increased up to 637 mg kg^?1 dietary Mg and then levelled off beyond this level. However, Ca and P contents seem to be inversely related to dietary Mg. Dietary Mg levels higher than 473 mg kg^?1 significantly decreased Zn and Fe contents in whole body and vertebrae. Broken‐line analysis indicated that 687 mg kg^?1 dietary Mg was required for maximal tissue Mg storage, as well as satisfied for the optimal growth.     

Dietary calcium requirement and effects on growth and tissue calcium content of juvenile grass carp (Ctenopharyngodon idella)

A growth trial was conducted to estimate the optimum concentration of dietary calcium (Ca) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (4.52 ± 0.02 g) were fed diets containing graded levels (2.75, 4.51, 6.24, 7.99, 9.66 and 11.5 g kg^?1) of Ca for 8 weeks. Weight gain, feed efficiency and protein efficiency ratio were linearly increased up to the 7.99 g kg^?1 dietary Ca and then maintained stable beyond this level (P < 0.05). Dietary Ca levels higher than 7.99 g kg^?1 significantly increased the ash contents of whole body, vertebrae and scales. Ca contents in whole body, vertebrae and scales were linearly increased up to the 7.99 g kg^?1 dietary Ca and then maintained stable beyond this level (P < 0.05). In contrast, dietary Ca levels higher than 9.66 g kg^?1 significantly decreased Mg contents in whole body, vertebrae and scales. Dietary Ca levels higher than 7.99 g kg^?1 significantly increased plasma alkaline phosphatase activity. However, plasma Ca, P and Mg contents were not significantly affected by dietary Ca supplements (P > 0.05). Polynomial regression analysis indicated that 10.4 g kg^?1 dietary Ca was required for maximal tissue storage and mineralization as well as optimal growth.     

Molecular cloning of fatty acid synthase from grass carp (Ctenopharyngodon idella) and the regulation of its expression by dietary fat level

The fatty acid synthase (FAS) gene was cloned from liver of grass carp (Ctenopharyngodon idella) by degenerate oligonucleotide primed PCR. The obtained cDNA fragment was 683 bp, which encoded 227 amino acids. Then, grass carps with initial body weight of (134.89 ± 12.12) g were fed diet supplemented with 0, 20, 40 g kg^?1 fat to investigate the impacts of dietary fat levels on growth, liver FAS enzyme activity and mRNA expression. After 8 weeks feeding, final body weights of the three groups were 344.11, 347.23 and 373.02 g. Compared with control group (0 g kg^?1), growth rate of 40 g kg^?1 fat group was increased by 14.03%, and feed conversion rate decreased by 11.32% (P < 0.05), liver FAS enzyme activity of 20, 40 g kg^?1 fat groups were reduced by 33%, 64% (P < 0.05), and FAS mRNA expression level reduced by 18%, 74% (P < 0.05), respectively. Results above showed that 40 g kg^?1 fat addition can significantly improve growth performance of grass carp. Liver FAS activity and mRNA expression tended to be inhibited by the increasing dietary fat level. Fat containing high levels of polyunsaturated fatty acids had strongly inhibitory effects on liver FAS activity and gene expression.