草鱼HSP90基因cDNA序列克隆及其组织表达差异

根据斑马鱼HSP90(Heat shock protein)序列(NM_131328)设计并合成一对引物,提取草鱼肝脏组织总RNA,RT-PCR扩增获得草鱼HSP90基因cDNA部分序列596 bp,获得GenBank登陆号为FJ517554.将测序结果利用DNAman软件与其他几种已知序列的鱼进行比对,结果表明,草鱼HSP90与斑马鱼、鲤、鲋的同源性依次为90%、89%、92%.同时利用半定量(Semi-quantitative,SQ)RT-PCR技术检测HSP90在草鱼脂肪、肌肉、肠、脑、粘液、性腺、鳔、肝脏、心脏、脾脏、鳃、鳍12个组织的表达差异.结果表明,HSP90在草鱼除了鳍外的其他11个组织中均检测到表达,其中在心脏中表达最高,但与鳃、性腺、脑、脾脏中的表达无显著差异(P>0.05),在脂肪、粘液、鳔组织中的表达显著低于其他几种组织(P<0.05),在肝脏、肌肉与肠中的表达无显著差异(P>0.05).     

长鳍吻免疫因子基因cDNA部分序列克隆及其组织表达差异

根据鲤科鱼类同源序列设计并合成了长鳍吻(Rhinogobio ventralis)应激因子HSP70、抗体IgM、抑炎性因子IL-10和促炎性因子IL-1b基因特异引物,以长鳍吻皮肤组织总RNA为模板,RT-PCR扩增获得长度分别为412、463、520和217 bp的上述4种免疫因子基因cDNA部分序列。同时通过RT-PCR比较患小瓜虫病长鳍吻和对照组长鳍吻皮肤和肠道组织中各免疫因子的表达差异,结果显示:在皮肤组织中,IgM和IL-10在感染组鱼体中表达量显著高于对照鱼(P<0.05),HSP 70和IL-1b的表达则没有差异;在肠道组织中,IgM、IL-10和IL-1b在感染组鱼体中表达量显著增高,HSP 70的表达量没有差异。本研究首次对长鳍吻的免疫因子进行了分析,指出免疫因子IgM和IL-10在鱼体中免疫应答反应中较为灵敏,为养殖过程中长鳍吻应激监测方面奠定了理论基础。     

草鱼NADPH氧化酶2个调节亚基cDNA的克隆及表达特征

为了解草鱼(Ctenopharyngodon idellus)NADPH多酶复合体的基因结构及其在机体的防御体系中的功能,利用RT-PCR结合RACE-PCR的方法,克隆草鱼NADPH氧化酶的2个调节亚基p40phox和p47phox的cDNA,对其编码的氨基酸序列进行了同源性比较和功能域分析,同时对这两个亚基在草鱼不同组织中的差异性表达进行分析。结果显示:p47phox亚基cDNA序列全长为1 589 bp,开放阅读框长度为1 233 bp,编码410个氨基酸;p40phox亚基cDNA序列全长为2 103 bp,开放阅读框为1 068 bp,编码355个氨基酸。这两个亚基编码的氨基酸序列与其他鱼类的同源性在68%~96%,具有其它鱼类类似的PX,SH3,PB1和PC功能域。组织差异性表达分析结果表明:2个调节亚基在草鱼胸腺、心脏、头肾、鳃、肠、肝、肾、脾和皮肤组织中均有表达,在不同组织中的表达强度略有差异,在心脏、胸腺中表达水平最高,在肝脏中表达水平较低,但是不同组织之间的表达水平差异不显著(P>0.05)。     

草鱼干扰素3蛋白多克隆抗体制备及其应用

为探讨干扰素3(Interferon 3,IFN3)在抗病毒免疫应答中的作用,以草鱼(Ctenopharyngodon idella)巨噬细胞cDNA为模板,PCR扩增IFN3成熟肽基因序列,制备草鱼IFN3蛋白的多克隆抗体,同时研究了IFN3在草鱼不同免疫组织中的蛋白表达,以及草鱼呼肠孤病毒(grass carp reovirus,GCRV)感染草鱼肾细胞系(Ctenopharyngodon idella kidney,CIK)后不同时间点的表达。结果显示,细菌表达的重组IFN3大小约为45 kD,主要以包涵体形式存在;抗体效价约为1∶3 200,制备的多克隆抗体既能识别原核表达的重组蛋白,也能识别个体水平上和细胞水平上的内源蛋白。草鱼主要免疫组织中,肝胰腺和皮肤检测到相应条带。CIK细胞感染病毒后12 h开始检测到IFN3蛋白,随感染时间的延长,IFN3蛋白表达量有所增加。蛋白水平上检测IFN3的表达,为深入研究草鱼的抗病毒免疫机制奠定了基础。     

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。     

热休克蛋白70(HSP70)基因在中国对虾染色体上的定位

以热休克蛋白70(HSP70)基因为探针,利用荧光原位杂交(FISH)的方法,将其初步定位在中国对虾(Fenneropenaeus chinesis)染色体上。观察150组精巢细胞染色体,有111组染色体有3个杂交信号,占所观察的74％,由此得出,热休克蛋白70基因(HSP70)很可能存在于中国对虾减数分裂细胞3对染色体的3个位点上。本研究首次将功能基因定位在了对虾染色体上,为中国对虾的遗传物理图谱的构建提供了有效可行的方法。     

草鱼朗格汉斯细胞的组织分布及其特征基因CD207的功能分析

为了研究草鱼(Ctenopharyngodon idella)体内朗格汉斯细胞(Langerhans cells, LCs)的分布及其特征性基因CD207的功能,利用免疫荧光(immunofluorescence, IF)、蛋白质印迹、实时荧光定量PCR(quantitative Real-time PCR,qPCR)探究草鱼LCs在相关免疫组织的表达情况及其特征性基因CD207在感染中的调节作用。免疫荧光结果显示,LCs主要在草鱼的头肾,皮肤的表皮和真皮,肠道固有层和鳃的鳃小片中分布。蛋白质印迹和qPCR结果显示,在LPS、PolyI:C处理头肾白细胞后,CD207的mRNA和蛋白相对表达水平均有显著上调。结果表明在抗原入侵机体后,草鱼LCs可以通过上调CD207的表达,在抵抗病原感染过程中发挥重要功能。     

合浦珠母贝热休克蛋白hsp70基因的克隆与表达分析

采用同源克隆和RT-PCR技术对合浦珠母贝(Pinctada fucata)热休克蛋白hsp70基因进行了克隆和表达分析。获得cDNA全长序列2 365 bp,其中3’非编码区域(UTR)为318 bp,5’UTR为88 bp,开放阅读框(ORF)为1 959 bp,编码652个氨基酸,分子量约为71.39 kD,理论等电点为5.22,并含有3个HSP70家族的签名序列IDLGTTYS、DLGGGTFD和EEVD。同源性分析表明,合浦珠母贝HSP70的氨基酸序列与太平洋牡蛎(Crassostrea gigas)等双壳贝类的相似性高达86%以上,基于氨基酸序列的聚类分析表明,合浦珠母贝与牡蛎属种类亲缘关系最近。高温、高盐刺激后,半定量RT-PCR检测发现hsp70基因的表达明显增加,高温刺激的表达量高于高盐刺激,高温刺激组不同组织的表达量由大到小依次为鳃、消化腺、外套膜、肌肉、性腺,高盐刺激组不同组织的表达量由大到小依次为鳃、外套膜、肌肉、消化腺、性腺,表明HSP70参与了机体对刺激的应答过程。该基因的克隆为进一步深入研究合浦珠母贝的抗逆机理及其遗传改良奠定了重要基础。     

长鳍吻(鳍)免疫因子基因cDNA部分序列克隆及其组织表达差异

根据鲤科鱼类同源序列设计并合成了长鳍吻(鳍)(Rhinogobio ventralis)应激因子HSP70、抗体IgM、抑炎性因子IL-10和促炎性因子IL-1b基因特异引物,以长鳍吻(鳍)皮肤组织总RNA为模板,RT-PCR 扩增获得长度分别为412、463、520和217 bp的上述4种免疫因子基因cDNA部分序列.同时通过RT-PCR比较患小瓜虫病长鳍吻1715和对照组长鳍吻(鳍)皮肤和肠道组织中各免疫因子的表达差异,结果显示:在皮肤组织中,IgM和IL-10在感染组鱼体中表达量显著高于对照鱼(P<0.05), HSP 70和 IL-1b的表达则没有差异;在肠道组织中,IgM、IL-10和 IL-1b在感染组鱼体中表达量显著增高,HSP 70的表达量没有差异.本研究首次对长鳍吻(鳍)的免疫因子进行了分析,指出免疫因子IgM和IL-10在鱼体中免疫应答反应中较为灵敏,为养殖过程中长鳍吻(鳍)应激监测方面奠定了理论基础.     

草鱼PPARα和PPARγ基因的克隆与组织表达差异

根据GenBank上登录的过氧化物酶体增殖物激活受体s(Peroxisome proliferator activated receptor,PPARs)基因序列设计2对引物,提取草鱼肝脏组织总RNA,经RT-PCR扩增获得了PPARα 624bp和PPARγ438 bp.并利用半定量RT-PCR分析T草鱼PPARα和...     

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。     

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.     

长江草鱼群体MHC Class ⅡB的多态性和进化分析

克隆及测序草鱼(Ctenopharyngodon idella)长江3个群体的主要组织相容性复合体(MHC)Class II B基因编码β1和β2区的第2和第3个外显子及两个外显子之间的内含子,分析了草鱼MHC的进化模式和种群遗传结构。结果显示:实验共定义了34个等位基因,每条序列包括长为130~136 bp的第2个外显子,长为218 bp的第3个外显子以及长81~371 bp的内含子。序列分析揭示,第2个外显子有106个核苷酸变异位点(78%)和40个氨基酸变异位点(88%),而第3个外显子有100个核苷酸变异位点(45%)和41个氨基酸变异位点(56%),β1变异要大于β2区。用β1和β2区序列分别构建的邻接(NJ)系统树均显示5个具有高支持率的谱系,结合序列变异特点和内含子长度,推测草鱼至少存在5个MHC Class II座位。分别计算β1的肽结合位点(PBR)、非肽结合位点及β2的非同义替换率(dN)和同义替换率(dS),PBR的dN/dS为2.03(P<0.05),非肽结合位点和β2则小于1,表明草鱼MHC受到歧化选择作用。根据等位基因在群体中的分布频率作分子方差分析(AMOVA),得出FST为0.37%,提示长江草鱼MHC没有遗传分化。     

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.     

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 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 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.     

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。     

草鱼肠道芽孢杆菌的鉴定及产酶能力分析

从100 g左右的健康草鱼(Ctenopharyngodon idellus)肠道中分离出3株疑似芽孢杆菌(Bacillus)菌株(G1、G2、G3),并通过对其进行生理生化特征、16S rDNA全序列和产酶能力的分析,筛选出1株高产酶能力的益生菌株.结果显示:G1、G2和G3菌株与枯草芽孢杆菌在16S rDNA序列相似性≥99％的水平上聚为同一分支,结合形态观察和生理生化特征,最终鉴定G1、G2和G3菌株均为枯草芽孢杆菌(Bacillus subtilis);以水解圈直径/菌落直径比值评定了G1、G2和G3菌株的产纤维素酶、淀粉酶和蛋白酶的能力,其中G1菌株产纤维素酶、淀粉酶和蛋白酶能力(比值分别为2.9、2.2和3.3)均高于G2、G3菌株,具有作为益生菌的潜力.