虹鳟免疫诱导型基因Cathelicidin2启动子功能分析

本研究通过实时荧光定量PCR实验对虹鳟(Oncorhynchus mykiss)Cathelicidin2(Cath2)基因的转录模式进行了分析。结果显示,该基因在鳃、头肾等与机体免疫防御功能密切相关的组织内转录,在细菌和病毒感染后,转录水平均显著升高。对基因上游调控序列进行启动子和转录因子结合位点预测,发现该启动子具有真核生物典型的TATA盒和CAAT盒结构,基因上游直至第一内含子区域内,密集存在多个免疫相关转录因子结合位点,其中,2个核因子κB(Nuclear factor kappa B,NFκB)预测结合位点均位于核心启动子正链区域。在草鱼(Ctenopharyngodon idellus)肾组织细胞系内,绿色荧光蛋白和萤火虫荧光素酶基因都能够在该启动子驱动下表达,表明其具有启动子活性,且启动子活性在受到免疫诱导后增强,包括细菌脂多糖(Lipopolysaccharides,LPS)模拟的细菌感染和聚肌胞苷酸(Polyinosinic polycytidylic acid,Poly I:C)模拟的病毒感染。双荧光素酶报告基因检测显示,启动子活性在与NFκB转录因子表达时,增强至4.39倍,证明Cath2基因受NFκB通路调控。研究表明,虹鳟Cath2基因能够在多种免疫刺激诱导下表达,其启动子可以应用为免疫诱导型的基因工程元件,驱动外源免疫基因在鱼体内适时表达,抵御外界病原感染,同时,避免非必要条件下的过度表达形成生长负担。     

鲤免疫应答相关基因的克隆与鉴定

为研究鲤(Cyprinus carpio L.)白细胞免疫应答相关的分子机理,以体外培养的鲤外周血白细胞为实验材料,用荧光标记的mRNA差异显示(FluoroDDRT-PCR)技术,研究丝裂原(50μg/mL LPS、50μg/mL PHA和50μg/mL ConA)在刺激白细胞4、12和24 h内诱导白细胞免疫应答相关基因的mRNA表达差异,共获得92个差异片段,其中87个片段有再扩增产物,再扩增率为94.6%;将差异片段克隆,经PCR鉴定,获得81个阳性克隆,鉴定率为93.1%;差异片段序列同源性功能分析结果表明,本研究共获得3个免疫应答相关的cDNA克隆,它们分别编码鲤的蛋白酶体激活因子PA28α亚基、翻译延伸因子(EF-1α)和基质金属蛋白酶13(Mmp13)部分序列,为进一步研究这些差异表达基因在鱼类免疫中的作用机制奠定了基础。     

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。     

鲤WISP基因家族的克隆、表达及系统进化树的构建

Wnt1诱导分泌蛋白(WISP)基因家族与CYR61、CTGF、NOV基因共同构成了CCN家族。研究通过鲤基因组序列与斑马鱼WISP基因编码区全序列的比对获得WISP1a、WISP1b、WISP2、和WISP3等4条序列。经过克隆、测序、比对拼接得到其开放阅读框,分别是1 089、1 077、1 038和1 026 bp,它们都是由5个外显子和4个内含子构成,分别编码362、358、345和341个氨基酸。分子系统学分析表明,鲤4个WISP基因具有高度同源性,其中WISP1a、WISP1b和WISP2均含有4个模块,WISP3缺少第2个模块。通过RT-PCR检测WISP基因在鲤组织中的表达,结果表明,WISP1a鲤在13个组织中均有表达,皮肤中表达最高,脾、肠、卵巢次之,其他组织中表达较低。WISP1b在精巢、脑、皮肤、卵巢中表达较高。WISP2在血液、鳃中表达较高。WISP3在血液、脑、肝中表达较高。实时荧光定量PCR法分析WISP基因在鲤胚胎发育时期的表达,结果表明,除WISP3外,WISP1a、WISP1b和WISP2在前期表达较高,36 h表达最低,随后逐渐升高至第6天开始下降。     

鲤胰岛素样生长因子结合蛋白-2和-3基因启动子克隆与序列分析

胰岛素样生长因子(insulin-like growth factors,IGFs)系统对鱼类的生长和繁殖有着重要的作用。利用PCR方法克隆获得了鲤胰岛素样生长因子结合蛋白(insulin-like growth factor binding protein,IGFBP)-2和-3基因的上游启动子部分序列,长度分别是1142bp和1067bp。Igfbp-2启动子区域没有TATA框和CAAT框。同时,Igfbp-3中只含有TATA框,但没有CAAT框。研究结果表明,二者启动子不具备典型的启动子特征,同时,在二者启动子上还发现了cAMP应答元件和肝细胞核因子结合位点,以及Pit-1、Oct-1、RXR和GR等结合位点。研究认为,鲤Igfbp-2和-3基因的表达受到潜在的多因子调控。     

大黄鱼slitrk3基因启动子克隆及转录调控分析

神经突触相关蛋白Slitrk3具有调节抑制性突触发育的作用。研究大黄鱼(Larimichthys crocea)神经突触黏附分子slitrk3基因的转录调控机制,可为解决大黄鱼养殖面临的生长、应激及抗逆等问题提供新思路。通过生物信息学方法对大黄鱼及其他物种的Slitrk3氨基酸进行多重序列比对和系统进化树分析,并预测大黄鱼slitrk3基因启动子区域相关的调控元件,采用双荧光素酶报告基因系统检测大黄鱼slitrk3基因启动子区域的转录活性。生物信息学分析结果显示,Slitrk3氨基酸序列在鱼类中具有较高的保守性;大黄鱼slitrk3基因启动子区域预测存在2个潜在的转录起始位点、2个CpG岛以及Sp1、GR、C/EBPα和C/EBPβ等多种转录因子结合位点。双荧光素酶报告基因系统结果显示,大黄鱼slitrk3基因启动子区域的-1 970—-1 614 bp、-1 210—-667 bp存在正调控元件,-1 614—-1 210 bp、-667—-376 bp、-376—-147bp存在负调控元件,推测-147—+16 bp为核心启动子。     

镉胁迫下鲤血液免疫指标的变化

水体中镉含量超标会引起鱼体免疫反应,影响鱼类免疫相关酶的活性以及免疫因子的表达。通过对鲤(Cyprinus carpio)幼鱼进行镉(Cd)暴露,检测了血液中非特异性免疫相关指标。结果显示,0.05 mg/L和0.5 mg/L的镉可以引起血清溶菌酶活性的显著升高;0.5 mg/L的镉导致血液白细胞吞噬百分比的显著降低;0.005 mg/L、0.05 mg/L和0.5 mg/L镉均导致鲤血清中酸性磷酸酶活性的显著升高;0.05 mg/L和0.5 mg/L镉引起血清中碱性磷酸酶活性的显著降低;0.005 mg/L、0.05 mg/L和0.5 mg/L镉暴露组的血细胞呼吸爆发活性与对照组相比极显著降低。研究发现镉对鲤幼鱼具有免疫毒性,酸性磷酸酶和血细胞呼吸爆发活性发生了显著变化,说明酸性磷酸酶和血细胞呼吸爆发活性对镉较为敏感,可以作为水环境中低浓度镉的标志物。镉暴露组的血细胞爆发活性的显著降低,证明镉的免疫毒性与体内氧化应激相关。     

投喂阿维拉霉素对鲤免疫应答的影响

将阿维拉霉素按照一定的浓度添加到饲料中,投喂经注射接种福尔马林灭活的嗜水气单胞菌(Aerom onashydrophila)菌苗的鲤,于免疫后的第7 d、14 d、21 d、28 d和35 d检测供试鲤血液中的白细胞吞噬活性、血清中溶菌酶活性、抗体效价、补体C3含量及免疫保护率(RPS),探讨了阿维拉霉素对受免鲤免疫应答的影响。结果表明,饲料中添加阿维拉霉素(4 mg/kg和8 mg/kg),投喂较长时间(≥28 d)会降低受免鲤的血液白细胞吞噬活性、血清中的溶菌酶活性和抗体效价,减少受免鲤血清中补体C3的含量,减弱鲤抵抗嗜水气单胞菌人工感染的能力,每公斤饲料中添加8 mg的阿维拉霉素其免疫抑制作用更强。     

易捕鲤F_3与亲本大头鲤一龄鱼种生长性能对比试验

在环境相同的池塘中对易捕鲤F3及亲本大头鲤的一龄鱼种进行生长对比试验,结果:易捕鲤F3的平均体重和绝对增重率都显著高于大头鲤;肥满度亦显著大于亲本大头鲤。以上结果表明,杂交选育的后代易捕鲤F3生长性能好于亲本大头鲤。     

易捕鲤F3与亲本大头鲤一龄鱼种生长性能对比试验

在环境相同的池塘中对易捕鲤F3及亲本大头鲤的一龄鱼种进行生长对比试验,结果:易捕鲤F3的平均体重和绝对增重率都显著高于大头鲤;肥满度亦显著大于亲本大头鲤。以上结果表明,杂交选育的后代易捕鲤F3生长性能好于亲本大头鲤。     

鲤Rhbdd3蛋白的抗病毒功能

Rhbdd3(Rhomboid domain-containing protein 3)蛋白在哺乳动物天然免疫中发挥了重要作用,但水生动物中rhbdd3基因的确定序列及Rhbdd3蛋白的功能均尚未见报道。为研究鲤(Cyprinus carpio)的Rhbdd3蛋白在鱼类细胞中的功能,探讨其过表达对鱼类病毒感染的影响,本研究通过PCR扩增得到了鲤rhbdd3基因的编码序列,并将其克隆至pCI-neo载体上,构建了真核表达质粒pCI-rhbdd3。pCI-rhbdd3转染鲤上皮瘤细胞EPC(epithelioma papulosum cyprinid)和鲑囊胚细胞CHSE-214(chinook salmon embryo)后利用制备的特异性抗体进行Western blot,检测Rhbdd3蛋白的表达情况,并利用CCK-8试剂检测其过表达对细胞增殖的影响。转染后分别进行鲤春病毒血症病毒(SVCV)和传染性胰腺坏死病毒(IPNV)的感染实验,并利用间接免疫荧光、Western blot和RT-qPCR方法检测Rhbdd3过表达对SVCV和IPNV增殖的影响。结果显示,pCI-rhbdd3转染后Rhbdd3蛋白在EPC和CHSE-214细胞中得到了过表达,且Rhbdd3蛋白的过表达能显著抑制SVCV和IPNV的复制,但不影响两种细胞的正常活性。本研究为鱼类广谱抗病毒药物的开发提供了新的实验依据,也为鱼类抗病毒新品种的培育奠定了重要基础。     

鲤硒缺乏的病理学研究

将健康幼鲤360尾设置3个相同平行处理,每个处理用鱼120尾,随机分为四组,分别投以0 mg/kg、0.15 mg/kg、0.30 mg/kg和0.45mg/kg硒水平的饲料,各试验组的发病率和死亡率与日粮中硒含量高低呈负相关,其发病率分别为46.7%、33.3%、13.3%和0%,死亡率分别为26.7%、16.7%、6.7%和0%。病鱼出现特征性的“瘦背病”和脊柱弯曲等症状。组织学上最突出的变化表现为营养性肌病、营养性肝病、胰腺的变性、坏死等多器官组织的退行性变化。超微结构上,骨骼肌纤维、肝细胞和肾小管上皮细胞线粒体肿胀,嵴断裂,甚至发生溶解,整个线粒体呈囊泡状。硒缺乏幼鲤血液中谷草转氨酶（AST）、谷丙转氨酶（ALT）活性升高,谷胱甘肽过氧化物酶（GSH-Px）、过氧化物歧化酶（SOD）活性降低,丙二醛（MDA）含量升高。     

锦鲤microRNA-137系统进化、靶基因验证及表达分析

为了探究microRNA-137(miR-137)与锦鲤体色形成的关系,实验对已报道的14种鱼类miR-137前体序列(precursor miR-137, pre-miR-137)进行比对,利用最大似然估计法(Maximum Likelihood Estimate, MLE)构建系统进化树,同时荧光定量PCR (quantitative realtime PCR, qRT-PCR)分析其在体色发生阶段和不同组织中的相对表达水平,并预测其靶基因,随后对靶基因进行GO和KEGG分析,最后通过荧光素酶实验验证miR-137-3p与靶基因的靶向关系,并分析miR-137-3p与靶基因mRNA的表达变化。结果显示,锦鲤与鲤的pre-miR-137序列相似度最高,且pre-miR-137序列在所有鱼类中具有较高的保守性。对miR-137靶基因进行GO和KEGG分析,多个靶基因富集在色素沉积、色素细胞分化等信号通路。定量结果显示,锦鲤出膜后11 d(day post hatching, dph) miR-137-3p表达量达到最高,随后显著降低;miR-137-3p在锦鲤不同组织中均有表达,在眼和肌肉中表达量较高,在皮肤和鳍条等色素细胞存在的组织中也有较高表达,且白色组织表达量显著高于红色组织。荧光素酶实验结果显示,miR-137-3p可结合在mitfa 3′-UTR上并抑制其表达,但对sprb并无显著抑制作用;miR-137-3p与mitfa和sprb在不同发育阶段存在显著负相关,但在组织中不存在相关性。本研究发现,miR-137在鱼类进化过程中高度保守,与锦鲤体色形成具有一定的相关性,且可靶向调控mitfa参与体色的形成,以上结果为进一步探讨miR-137在锦鲤体色形成中的作用提供基础数据。     

Top-crossing with evaluation of slaughtering value in common carp (Cyprinus carpio L.) offspring

A three-year test of growth performance andoffspring survival from top-crossing of commoncarp hybrids in low altitude (350 m above thesea level, middle European climate) and in highaltitude (750 m above the sea level, middleEuropean climate) was terminated by assessmentof slaughtering value of edible parts in therespective strains. A recently establishedHungarian synthetic mirror carp strain (HSM)was chosen for testing as maternal strain. TheHSM, as well as wild Amur carp (AC), Ropshacarp (ROP) and Tata carp (TAT) were used aspaternal strains.The evidential strain*altitude interactioneffect was demonstrated as significant forweight of fish (0.0005), fillet without skin(0.0001), index of highbackedness (0.005),index of head length (0.0404),Fulton's coefficient (0.0497),index of widebackedness (0.0315) andgonadosomatic index (0.0082). The interactioneffect was demonstrated as insignificantbetween altitude*sex and betweenstrain*sex.The comparative test of analysis of variance(ANOVA) revealed significantly (P < 0.05)higher weight of fish after killing on lowaltitude (1611.6 g) than on high altitude(1090.3 g), but processed body yield andpercentage of fillet without skin was notsignificantly different (64.0 and 34.2 for lowaltitude, 64.2 and 33.4 for high altitude). Thepercentage of processed body weight andpercentage of fillet without skin in analysisboth altitudes were significantly better infemales (65.5 and 34.6%) than in males (62.5and 33.00%), respectively. The highestsignificant differences in weight of fish afterkilling were found between HSM (890.8 g) andHSM × AC (1283.3 g) in high altitude. In lowaltitude, it was between HSM (1527.2 g) andHSM × AC (1706.9 g) and HSM × ROP (1693.0 g).     

Influence of nutrition on the lipid quality of pond fish: common carp (Cyprinus carpio) and tench (Tinca tinca)

Like marine fish freshwater fish are an important source of essential fatty acids for human nutrition. However, the fatty acid composition of pond fish can vary considerably and strongly depends on that of the ingested food. Investigations on the fatty acid composition of common carp (Cyprinus carpio) and tench (Tinca tinca) have shown that different methods of rearing and feeding cause substantial variations in the proportions of the n-6 and n-3 polyunsaturated fatty acids of these fish species. Carp reared on the basis of natural food in ponds exhibit high contents of n-6 as well as n-3 fatty acids in their muscle triacylglycerols. On the other hand carp fed supplementary wheat in ponds resulted in somewhat lower levels of these essential fatty acids. High amounts of n-3 fatty acids can be found in carp fed high-energy diets containing high levels of fish oil. Analogous results were obtained in experiments with tench reared under different nutritional conditions. While rearing on the basis of only natural food in ponds as well as feeding supplementary wheat yielded in similar levels of n-3 and n-6 polyunsaturated fatty acids, higher contents of n-3 fatty acids were recorded in tench fed pellets. High levels of n-3 polyunsaturated fatty acids in foodstuffs have positive effects on human health. Experiments with different cultured fish species proved that the fatty acid composition of the edible parts can be influenced by the diet. Therefore, a finishing diet with a suitable fatty acid profile can be used to improve the nutritional quality of fish products of farmed origin.     

Results of comparative growing test of all-female and bisexual population in two-year-old common carp (Cyprinus carpio L.)

The aim of this study was to verify whetherall-female carp populations grow faster thanbisexual ones. All-female mirror and scalypopulations were obtained by crossingmasculinizated females (neomales) of Northernmirror carp (M72) with regular females of M72and scaly Ropsa carp (ROP) purebreds,respectively. All-female and bisexual progenyof both mirror and scaly carps were rearedseparately for one year. However, controlgroups of a distinct phenotype of scalinesswere put in ponds with tested fish to make anadjustment for fish weight, which could becaused by environmental influence. Nodifferences were found after eight weeks ofrearing between all-female and bisexualpopulations, but, all-female mirror carppopulation (M72) gained 29.7% more weight(P < 0.0001) by the end of the first year thanthe bisexual (68.5 g ± 15.8 against 52.8 g± 13.9; mean ± S.D., respectively).Before beginning the second growing season,each group was marked and stocked together intriplicate ponds. Corrected weight after secondgrowing season (P < 0.0062) was 9.9% higher inall-female mirror carps (395.9 g ± 98.6)compared to the bisexual (360.2 g ± 89.0).No significant differences were found incorrected weights between scaly all-female andbisexual crossbreds (522.4 g ± 102.4 and518.3 g ± 95.8, respectively). Nodifferences in survival of all-female andbisexual populations were observed.     

中国鲤育种的主要方法、遗传解析及展望

水产种业是水产养殖业发展的基础,是渔业战略性、基础性核心产业,也是保障未来养殖业绿色、健康发展的核心竞争力。随着水产业全球化、市场化的发展,我国种业正面临着前所未有的机遇与挑战。特别是随着生物技术的快速发展,水产育种也由传统的选择育种和杂交育种,发展至细胞工程育种、分子标记辅助育种、全基因组选择育种、分子设计育种和基因组编辑等精准设计育种。水产动物重要经济性状的基础研究及其遗传改良技术的创建驱动着我国水产种业的蓬勃发展,截止2022年,通过全国水产原种和良种审定委员会审定并由农业农村部公告的水产新品种就有266个,其中鲤新品种数量最多,为31个,表明鲤育种工作卓有成效。本文重点回顾了我国鲤的种质和基因组资源现状,鲤的主要品种及其育种方法;简要介绍了鲤生长、抗病、体色、饲料转化率等经济性状关联的遗传研究进展,并由此提出了新时代背景下鲤种业的发展方向和措施建议,以期为我国鱼类育种提供借鉴。     

Effects of cyclic and regular feeding of a Quillaja saponin supplemented diet on growth and metabolism of common carp (Cyprinus carpio L.)

Fifteen common carp weighing 19.2±2.9 g were reared individually in chambers of a respirometer system which allowed feeding, and continuous measurement of oxygen consumption. The fish were divided into three groups of 5 each, and were given control feed (C group), control feed supplemented with 150 mg kg⁻¹ of Quillaja saponins (QS) (S150 group), and control and the saponin supplemented diet during alternate weeks (S150b group), for 56 days. Supplementation with QS at a level of 150 mg kg⁻¹ caused a significant (P<0.05) increase in metabolic growth rate, food conversion efficiency (FCE), protein productive value (PPV) and apparent lipid conversion (ALC) of carp, even as the metabolic rate was lower than the C group. The efficiency of energy utilisation indicated by parameters such as energy expenditure (EE), energy retention (ER) and amount of oxygen consumed per unit body mass gain (OPM) were also significantly (P<0.05) better in the S150 group. The performance of the S150b group was intermediate between the C and S150 group in this experiment. Possible reasons for the effects of dietary QS are discussed. It was concluded that there has to be continuous dietary supply of QS at the level of 150 mg kg⁻¹, for maximum positive effects of these substances on growth of common carp. This revised version was published online in August 2006 with corrections to the Cover Date.     

一种杂交鲤四种经济性状的QTL定位分析

以柏氏鲤和荷包红鲤抗寒品系杂交F21个家系的92尾个体为材料,用300个微卫星(SSR)标记构建了遗传连锁图谱,其长度为2 471.7 cM,标记间平均间隔为10.75 cM,在此基础上对体长(SL)、体厚(BT)、体高(H)和体质量(W)进行QTL定位分析.共检测到17个QTL区间分布于6个连锁群.4个体长的QTL中,位于LG5、LG7和LG19的QTL为显著水平(P＜0.05),位于LG32的QTL为极显著水平(P＜0.01),可解释表型变异率为5.14％～10.2％;4个体厚的QTL中,位于LG11(两个QTL)和LG32上的QTL为显著水平(P＜0.05),位于LG5的QTL达极显著水平(P＜0.01),可解释表型变异率为6.42％～ 8.43％;6个体高的QTL中,LG5,LG10,LG11,LG19,LG32上的QTL为显著水平(P＜0.05),LG7上达极显著水平(P＜0.01),可解释表型变异率为5.92％ ～ 8.95％;3个体质量的QTL中,位于LG5和LG7上的为显著水平(P＜0.05),可解释表型变异率为5.55％和8.36％,LG32上达极显著水平(P＜0.01),可解释表型变异率为6.44％.本研究采用了两个不同品系的杂交子代为作图群体,丰富了QTL研究群体的多样化,为今后不同鲤品系或品种间的QTL在全基因组水平上的比较和共性QTL的发掘等奠定基础,进而指导鲤分子育种.