团头鲂tf和tfr1a基因启动子克隆及转录调控分析

为探索鱼类转铁蛋白基因tf和转铁蛋白受体基因tfr1a的转录调控机制,本实验以团头鲂为研究对象,在其全基因组数据库中获取tf和tfr1a基因序列,对2个基因候选启动子区转录因子结合位点及CpG岛进行预测,通过PCR方法克隆得到tf和tfr1a基因近端启动子区不同长度片段,连接至pGL3-Basic/pEGFP-1载体,瞬时转染入Hela细胞,并采用双荧光素酶报告基因检测系统进行检测。结果发现,团头鲂tf基因启动子区无CpG岛位点,而tfr1a基因启动子区有2个CpG岛位点。成功构建9个tf和10个tfr1a不同长度启动子片段的重组质粒,经双荧光素酶报告基因系统检测发现,tf启动子核心区域为-268^+56 bp,且-1 308^-1 102 bp片段可能存在正调控该基因表达的转录因子结合位点;tfr1a启动子核心区域为-224^+48 bp,且+48^+92 bp可能存在抑制该基因转录的负调控元件,而-1 229^-1 219 bp区域可能存在促进tfr1a基因表达的正调控转录因子结合位点。     

Growth and ontogenetic development of digestive functionality in black Amur bream (Megalobrama terminalis)

The function of digestive physiology during ontogenetic development is essential to ensure high survival and growth rates. In order to evaluate the digestive physiological capacity of the black Amur bream (Megalobrama terminalis), changes of morphology and digestive enzyme activity (trypsin, lipase, amylase, pepsin, leucine aminopeptidase and alkaline phosphatase) in larvae were examined from hatching to 40 days after hatching (DAH). Results indicated that fluctuation patterns differed between the total and specific activities of the digestive enzymes. The total activities of these six enzymes gradually increased throughout the fish growth. The specific activity of trypsin peaked at 5 DAH and then decreased dramatically, while it increased remarkably again from 8 to 10 DAH and remained stable level after 20 DAH. Pepsin activity was first examined in M. terminalis at 15 DAH and gradually elevated towards the end of the experiment. The specific activity of lipase displayed obvious peaks at 5 and 20 DAH. For the amylase, its specific activity reached plateau at 4 DAH, underwent sharp decrease, and remained stable after 20 DAH. In addition, we found that the specific activity of alkaline phosphatase raised significantly from hatching to 5 DAH, and tended to keep slight fluctuation after 15 DAH. From the above, we concluded that the specific activities of digestive enzymes in the larvae varied constantly from 3 to 20 DAH and turned relatively stable after 20 DAH. The present study provides effective information that is useful to improve the seedling cultivation and the technology for healthy breeding.     

团头鲂(Megahbrama amblycephala) Caspase 9 基因全长cDNA的克隆及在氨氮胁迫下的表达分析

为了解团头鲂(Megahbrama amblycephala) Caspase 9基因序列特征及其在氨氮胁迫过程中的作用,应用末端快速扩增(Rapid amplification of cDNA ends,RACE)技术克隆得到团头鲂Caspase 9基因全长1613 bp的cDNA序列,包括185 bp的5 '末端非翻译区(Untranslated regions,UTR)、96 bp的3'UTR、1332 bp的开放阅读框(Open Reading Frame,ORF).氨基酸多序列比对显示,平均同源性为77.74％,表明团头鲂Caspase9基因具有较高的保守性;系统进化分析显示,该基因氨基酸序列与其他鱼类Caspase 9聚为一支,并与锦鲤(Cyprinus carpio) Caspase 9亲缘关系较近;荧光定量PCR结果显示,Caspase 9基因在团头鲂各组织中均有表达,在脑中表达量最高,在肌肉中表达量最低,同时,在胁迫和恢复过程中,该基因在肝和脑中的表达规律相似,均在胁迫期间出现表达量上调,整体呈类似波浪形表达谱.研究结果表明,氨氮胁迫下,Caspase 9基因参与团头鲂的免疫防御,并与细胞凋亡分子过程有关.本研究结果可为进一步了解团头鲂应答氨氮胁迫分子机制提供理论依据.     

Essential amino acids supplementation to practical diets affects growth,feed utilization and glucose metabolism‐related signalling molecules of blunt snout bream (Megalobrama amblycephala)

Six diets were formulated to investigate the success of fish meal (FM) replacement by plant proteins; diet 1 reflected a commercial feed (8% FM), diet 3 contained 4% FM, and diet 5 was devoid of FM. Whereas, diet 2, diet 4, and diet 6 reflected diet 1, diet 3, diet 5, respectively, and supplemented with essential amino acid (EAA). At the end of 8‐week trial, there was no significant difference in survival rate. Significantly higher final weight, weight gain rate, and specific growth rate were recorded in the group fed diet 2 compared with the other treatments (except diet 4) (p < 0.05). Feed conversion ratio of fish fed diet 2 was significantly lower than those fed diets 1, 3, and 5 (p < 0.05). The lowest feed intake and highest protein efficiency rate were found in fish fed diet 2 (p < 0.05). There was no significant difference in whole body compositions between treatments. Plasma aspartate transaminases, alanine aminotransferase, and glucose were significantly affected by dietary treatments (p < 0.05), while plasma protein and albumin contents were not influenced by the treatments. The relative expression of target of rapamycin (TOR) and phosphatidylinositol 3‐kinase, regulatory subunit 1 (alpha) (PIK3R1) in fish fed diet 3 (4% FM) were significantly down‐regulated compared with those fed diet 6 for TOR and diets 4 and 6 for PIK3R1 (p < 0.05). Insulin receptor substrate 1 (IRS‐1) and janus kinase 3 (JAK3) expressions were fluctuated, with the higher levels in fish fed diets 4 and 6. In conclusion, the findings of this study indicate that plant protein mixture supplemented with EAA could be used to substitute FM in practical diet for Megalobrama amblycephala.     

溶解氧对团头鲂耐低氧新品系F5代的鳃组织形态及各组织酶活性的影响

为研究不同浓度溶解氧(DO)对团头鲂(Megalobrama amblycephala)耐低氧新品系F5代鳃组织形态以及各组织酶活性的影响,本研究将团头鲂在低氧[DO为(1.7±0.2) mg/L]和高氧[DO为(19.3± 0.5) mg/L]条件下分别处理0、4和7 d,恢复常氧[DO为(7.8±0.3) mg/L] 7 d后,通过石蜡切片观察鳃组织的形态,并测定了鳃、肝胰腺、肠道、肌肉中过氧化氢酶(CAT)、琥珀酸脱氢酶(SDH)及乳酸脱氢酶(LDH)活性和丙二醛(MDA)含量。石蜡切片结果显示,随着低氧处理时间的延长,团头鲂新品系鳃丝的层间细胞团体积减小,鳃小片表面积增加,恢复常氧7 d后又有所恢复。高氧条件下,层间细胞团体积增大,鳃小片表面积减小,恢复常氧7 d后也会恢复。酶活性检测结果显示,在低氧和高氧2种条件下,随着处理时间的延长,CAT活性和MDA含量在各组织中的变化无明显规律,但均有显著差异(P<0.05)。低氧条件下,LDH活性显著增高,SDH活性显著降低(P<0.05)。高氧条件下,LDH活性显著降低,SDH活性显著增高(P<0.05)。本研究可为溶解氧对团头鲂的鳃组织以及各组织酶活性的影响提供基础资料,并为团头鲂新品系的养殖与选育奠定基础。     

团头鲂池塘养殖生态系统晒塘阶段温室气体排放通量分析

为探讨团头鲂池塘养殖生态系统晒塘阶段温室气体的排放规律及综合增温潜势,采用静态暗箱——气相色谱法对团头鲂池塘养殖生态系统晒塘阶段温室气体(CO2,CH4,N2O)的排放进行原位测定。结果显示,团头鲂池塘养殖生态系统晒塘阶段均表现为CO2,CH4和N2O的排放源,其中CO2排放通量达(86.72±12.46)g/m2,CH4排放量达(2.01±0.34)g/m2,N2O排放量达(7.44±0.98)mg/m2;在100 a的时间尺度上,团头鲂池塘养殖生态系统在晒塘阶段综合增温潜势为(157.28±24.31)g/m2,团头鲂池塘养殖生态系统温室气体减排空间较大。     

团头鲂jphs家族基因的克隆及表达分析

以团头鲂为研究对象,扩增获得Junctophilin(Jph)家族4个成员,jph1a、jph1b、jph2和jph3的cDNA编码序列,分别为2 031、2 016、2 358和2361 bp,分别编码676、671、785和786 aa。团头鲂jphs均由5个外显子和4个内含子组成,与大多数物种的JPHs基因结构相似;结构域预测显示出8个MORN和1个TM结构域,且蛋白多序列比对分析显示Jphs在进化上非常保守。基于qRT-PCR(quantitative real-time PCR)分析显示,这4个基因呈现出组织特异性表达,其中jph1a、jph1b和jph2主要在肌肉和心脏中表达,而jph3主要表达于心脏、血液和大脑。在早期发育过程中,jph1a、jph1b、jph2和jph3的mRNA表达模式类似,分别在原肠胚期、心跳期和25 dph(days post hatching)达到高峰。综上研究结果表明,鱼类jphs基因在心脏和肌肉等组织中发挥重要作用,并且其功能可能不同于哺乳动物。     

鄱阳湖、梁子湖、淤泥湖团头鲂池塘养殖对比试验分析

试验结果为：面积１６６６．８ｍ２的池塘，年生产商品鱼１０２１．８ｋｇ，其中团头鲂２４６．３ｋｇ，各水域团头鲂成活率均在９６％以上。试验结果证明三水域团头鲂有较强的适应性和抗病能力。但不同水域团头鲂的产量差异显著。     

团头鲂尾部神经分泌系统的超微结构及其在人工催产过程中的变化

在团头鲂尾部神经分泌系统的神经分泌细胞及其轴突中，可观察到三种不同类型的颗粒(即电子致密颗粒、电子致密度较低的颗粒和电子透明颗粒)。细胞质中线粒体、高尔基复合体和粗面内质网都比较发达，游离核糖体较多。在尾垂体的无髓神经纤维之间有时还可观察到桥粒结构。在性腺Ⅲ期至Ⅵ期，上述的细胞器变化不大，但三种颗粒的数量和分布却有明显的差别。根据团头鲂尾部神经分泌细胞和神经血管复合体超微结构的变化看来，人工催产有促进尾垂体电子致密颗粒中的分泌物向血管内释放的作用。     

基于SSR标记的鲌鲂“先锋2号”、团头鲂及黑尾近红鲌的群体遗传分析

从黑尾近红鲌发表的46对微卫星引物中筛选出的11对多态性较高的引物,分析团头鲂(♀)和黑尾近红鲌(♂)及其杂交后选育的F4代,即鲌鲂“先锋2号”三个群体的遗传多样性和遗传结构。研究结果显示:这三个群体之间的平均等位基因数分别为4.182、4.273、4.273;平均有效等位基因数分别为2.482、2.803和3.080;平均观测杂合度分别为0.712、0.380和0.507;平均期望杂合度分别为0.579、0.474和0.656;多态信息含量分别为0.508、0.414和0.572。鲌鲂“先锋2号”与母本团头鲂的遗传距离为0.2709,小于与父本黑尾近红鲌的遗传距离(0.5381),在亲缘关系树状图上先与母本聚为一支。鲌鲂“先锋2号”从母本中获得较多的遗传物质,较双亲有丰富的遗传多样性和更高的基因杂合度。研究结果将为研究团头鲂和黑尾近红鲌杂交后代的遗传多样性,探索鲌亚科鱼类属间杂交育种后亲本与子代的亲缘关系和遗传规律提供理论依据。