转基因超级鲤及子代的生长研究

用大麻哈鱼生长激素基因作转基因材料，获得了一批转基因鲤鱼和1尾“超级鲤”鱼。经分子检测证实外源基因整合到了受体鱼的基因组中并在体内表达出外源生长激素。连续多年的池塘生长实验表明，转基因鲤鱼的快速生长效应比正常鲤鱼明显，在试验鱼群体中最大个体体重比对照组群体中最大个体体重，分别提高了52．5％，46．0％，76．0％，而且由性成熟的超级转基因鲤鱼繁殖的F1代和F2代鱼，仍表现出快速生长的特性。     

人生长激素基因在团头鲂和鲤中的整合和表达

采用显微注射方法，将带有小鼠ＭＴ－１基因启动顺序与人生长激素ｈＧＨ基因顺序重组的线状ＤＮＡ片段，注入团头鲂和鲤的受精卵，获得成活的实验鱼。经斑点、Ｓｏｕｔｈｅｒｎ杂交、Ｎｏｒｔｈｅｒｎ杂交、放射免疫和酶联等方法检测，表明外源基因在受体鱼中得到整合、转录、翻译和表达，并具促生长效应。转基因雌鱼和雄鱼有性繁殖所获得的子鱼带有外源基因，表明外源基因能通过性细胞传递给子代，并仍具促生长效应。     

镜鲤体重相关分子标记与优良子代的筛选和培育

根据性状相关分子标记的QTL分析结果进行品种培育是分子标记用于水产育种研究的重要方向之一。本研究利用镜鲤与体重相关的3个基因座(HLJ302、HLJ338、HLJ343)作为快速生长的标记,筛选出一批镜鲤亲本进行繁殖,对得到的子代进行基因分型,并将基因分型结果与检测的最大10尾个体与最小10尾个体进行比较,结果表明最大10尾具有优势基因型为每尾1.7个,最小10尾具有优势基因型为每尾0.7个,生长速度快群体与生长速度慢群体在体重相关基因型的富集上呈现显著性差异。同时选出具有富集体重相关优势基因型的子代且生长速度快的群体一个,2008年生产出子二代。综合性状好生长速度更快的镜鲤新品系正在培育之中。     

应用RNAi技术构建的转基因鲤的检测分析

将构建携带H1启动子的肌肉生长抑制基因的真核表达载体,通过显微注射技术导入鲤受精卵核区附近,获得了一批具有RNAi表型的转基因鲤,PCR和分子杂交检测证实外源基因整合到受体鱼的基因组中,阳性率为32.78%;一龄鱼的生长实验表明,转基因鲤比普通鲤平均生长快0.99倍,其体高和体厚分别平均增长0.22和0.26倍,其中有31.82%的群体平均体厚是普通鲤的1.6倍。结果显示,该质粒表达的发夹环型dsRNA可以有效降解其转录产物,对阻抑肌细胞中同源基因的表达、鲤肌肉的再生能力增强起到了重要作用。这种抑制作用表现为鲤背部肌肉增厚、体质量增加,说明该基因经转录产生的双链RNA在鲤体内具有RNAi效应。RNAi技术为获得具有特殊功能的转基因鲤提供了新的手段。     

转生长激素基因工程鲤鱼研究

约10000余尾注射牛和羊生长激素基因的鲤鱼已被生产出来，并从中筛选出生长速度快且整合了外源基因的个体143尾，也证明外源基因可传递给子代同时子代也具有快速生长的特征。建立了把外源基因直接注入受精卵的方法，确定了注入外源基因的最佳时期。     

转生长激素基因鲤鱼的生长研究

本报导了生长激素基因对鲤鱼生长的影响。经池塘对照实验表明，转生长激素基因鲤鱼的快速生长效应比正常鲤鱼明显，而且由转生长激素基因阳性性成熟鲤鱼经人工催产繁殖的子一代群体中，仍表现出快速生长的特性。这一结果，为能获得一个转外源生长激素基因并具快速生长特性的遗传稳定的转基固鲤鱼核心群体提供了可靠的数据。     

转“大麻哈鱼”生长激素基因鲤对小鼠精子致畸性的影响

通过用转"大麻哈鱼"生长激素基因鲤鱼的肉浆给小鼠进行灌胃,研究转基因鲤对哺乳动物精子致畸的影响.结果显示,灌注非转基因鲤鱼和转"大麻哈鱼"生长激素基因鲤不同剂量实验组,对小鼠精子的畸形率没有显著的差异.证明食用转"大麻哈鱼"生长激素基因鲤对哺乳动物的精子发生是安全的,初步为转基因鲤的食品安全评价提供了理论依据.     

转基因技术及在水产养殖育种上的应用

近年来,作为分子生物学领域当中极具潜力的技术之一,转基因生物技术的应用在全球范围内以令人难以想象的速度发展起来。传统生物技术是借助自然选择或控制繁殖的方法进行物种内的基因改良,而转基因技术则是一种物种间的基因改良技术。它是指运用农杆菌介导、基因枪等方法,将外源基因导入到目的生物基因组中,使其获得人们所期望的新性状,并且外源基因可遵循遗传法则稳定遗传至子代,新性状在其子代稳定表达。自Palmiter1982年将大白鼠生长激素基因显微注射到小白鼠受精卵,获得比正常小鼠大1倍的“超级巨鼠”以来,世界各国科学家竞相开展转基因技术研究,并通过多种转基因方法在猪、牛、鸡、     

羊生长激素基因及表达产物对受体鲤鱼的促生长效应

两年的池塘生长对照实验证实了外源生长激素基因在受体鱼基因组整合及表达对部分受体鲤鱼有明显的促生长效应。实验组最大个体体重为对照组最大个体体重的1.47倍和1.15倍,从而预示转羊生长激素基因鲤鱼在速生鲤鱼品种培育方面有很好的应用前景,斑点杂交和Southern Blot杂交证实了外源基因确已整合在受体鱼基因组中,实验也表明外源基因在受体鱼基因组的插入对部分受体鱼的生长也有负效应。     

黄河鲤全同胞家系的微卫星标记亲子鉴定

本研究利用11个高度多态的微卫星标记对26个黄河鲤(Cyprinus carpio haematoperus)全同胞家系中的417尾体重较大的正常体色个体(黄河鲤较大个体)和52尾红色个体(红鲤表型个体)进行了亲子鉴定。11个微卫星座位的平均等位基因数、平均观测杂合度(H_o)、平均期望杂合度(H_e)和平均多态信息含量(PIC)分别为8.2、0.792、0.792和0.76。当双亲未知且置信度为95%时,11个座位的累积排除概率达99.79%。除29尾黄河鲤较大个体以外,388尾黄河鲤较大个体和52尾红鲤表型个体准确地找到了其父母本,实际鉴定率为93.82%。通过对子代数目超过20尾的7个黄河鲤家系生长性状的比较分析,本研究成功鉴定出子代生长性状优良的黄河鲤亲本组合及其家系,同时也鉴定出包含隐性红色基因的黄河鲤杂合亲本,为进一步选育表型和体色纯正且生长快速的黄河鲤新品种提供了理论依据和技术手段。     

建鲤生长激素促泌素受体1基因的特性及其与增重相关SNP位点的筛选

GHS-R基因在哺乳类为候选的肥胖和生长数量性状位点。本实验使用RT-PCR及PCR的方法,从建鲤(Cyprinus carpio var.jian)分离到2个jlGHS-R1s,称为jlGHS-R1a和jlGHS-R1b(分别简称1a和1b)。jlGHS-R1s的阅读框编码360个氨基酸,两者间相似性高达96%;另外还存在2个jlGHS-R1s的转录变体(分别简称1a’和1b’),选择性切割了翻译起始碱基后第490-680 nt的191 bp片段,该片段两端碱基分别为gt-ag,这种选择性切割导致翻译提前终止,仅翻译184个氨基酸,包括前面3个半跨膜区,这与已报道的罗非鱼等的保留内含子的转录变体不同。jlGHS-R1s的阅读框被1个内含子分隔,该内含子位于第260个氨基酸密码子的第1和第2个碱基之间,1a和1b的内含子长度分别为676 bp和885 bp。使用分段PCR在建鲤群体的1a和1b基因上共找到32个SNP位点,构建PCR-RFLP法对其中9个SNPs进行基因型检测,结果发现各种基因型在322尾建鲤中均有出现,但存在着明显的偏分布。与增重的相关分析表明,位点1a-I_C386T和1b-E1_G159T与雌雄鱼鱼种和成鱼阶段增重分别呈极显著(P<0.01)和显著(P<0.05)相关,CC型和GG型个体增重最快,另外有5个位点则与其中的某阶段或者某性别的增重相关,为了确定上述所得标记的适用性,选取其中4个标记在另外7个家系共610尾鱼中检测,结果 C386T和G159T还是与增重呈极显著(P<0.01)和显著(P<0.05)相关,可用于建鲤分子育种。     

红鲤生长性状的上位性遗传效应分析

运用加性-显性-上位性遗传模型及其分析方法,研究了兴国红鲤(Cyprinus carpiovar.singuonensis)、荷包红鲤(C.carpiovar.wuyuanensis)和瓯江彩鲤(C.carpiovar.color)的双列杂交子二代F2的体质量和10个形态性状(全长、体长、体高、体宽、头长、吻长、眼径、眼间距、尾柄长和尾柄高)的上位性遗传效应。结果表明,除体高外,其余性状均存在不同程度的加性×加性上位性方差,其中体质量、全长、体长、头长、吻长和尾柄长的加性×加性上位性方差达显著水平(P<0.05);这10项性状的上位性方差占总遗传变异量的百分率为:体质量11.4%、全长22.6%、体长14.0%、体宽8.3%,头长24.2%、吻长17.5%、眼径12.9%、眼间距23.1%、尾柄长5.4%和尾柄高20.1%;通过3种红鲤亲本的遗传育种效应预测发现,瓯江彩鲤的加性和上位性效应能显著增加后代的体质量、全长和体长,而兴国红鲤和荷包红鲤的加性和上位性效应能显著降低后代的体质量、全长和体长。上位性遗传效应在鱼类数量性状的遗传育种中具有较重要作用。[中国水产科学,2006,13(4):573-578]     

Effects of 3,5,3′-triiodo-L-thyronine (T3) and 6-n-propyl-2-thiouracil (PTU) on growth of GH-transgenic coho salmon, Oncorhynchus kitsutch

GH-transgeniccoho salmon (Oncorhynchus kitsutch) juveniles were fed diets containing 3,5,3-triiodo-L-thyronine (T₃; 30 ng/g fish) or 6-n-propyl-2-thiouracil (PTU; 20 ug/g fish), to assess the effect of these drugs on the physiology, growthand survival in comparison with untreated transgenicand non-transgenic salmon. After 84 days, food intake, feed efficiency, survival, growth, hepato-somatic index (HSI), viscera-somatic index (VSI), plasma L-thyroxine (T₄), T₃and growth hormone (GH) levels,and cranial morphological abnormalities were determined. Growth of transgenic salmon was significantly faster than the nontransgenic salmon,and was increased by exogenous T₃and reduced by PTU. Food intake of transgenic salmon was higher than that of the nontransgenic group, but was reduced by exogenous PTU administration. Food conversion efficiency of transgenic salmon was lower than that of nontransgenic salmon,and also was increased by T₃ but reduced by PTU in the transgenic fish. The survival rate in all transgenic groups was significantly higher than that of nontransgenic,and transgenic T₃and PTU treatment groups showed higher survivals than the transgenic-control group. The HSIand VSI of the transgenic fish were higher than the nontransgenic fish;and both parameters in the transgenic salmon were increased by PTU, but reduced by T₃. The plasma T₄ level in transgenic salmon was approximately 1.5-fold higher relative to the nontransgenic fish, whereas no difference was observed among the transgenic groups. Plasma T₃ levels in transgenic salmon were also approximately 2-fold higher relative to the nontransgenic fish. However, the plasma T₃ level in transgenic animals was increased by exogenous T₃ administration, but was reduced by exogenous PTU to that observed in nontransgenic salmon. The plasma GH level of transgenic fish was higher than that of the nontransgenic salmon,and the level was increased by the exogenous T₃, whereas exogenous PTU did not reduce significantly GH levels in transgenic salmon. Transgenic fish also displayed cranium, jawand opercular abnormalities typical of the effects of this gene construct incoho salmon, indicating that some imbalance in growth processes has been induced. However, these abnormalities (especially cranial disruptions) were diminished by administration of exogenous PTU. In conclusion, exogenous T₃and PTU treatments can induce hyperthyroidismand hypothyroidism, respectively,and have inverse effects on growthand skeletal abnormalities of transgenic salmon constitutively expressing GH.     

两种稻田养殖模式下青田田鱼与本地鲤鱼生长对比

2009年在贵州省遵义市湄潭县天城乡和黔东南州雷山县西江镇,比较了稻田传统养鱼模式和沟坑养鱼模式青田田鱼和贵州本地鲤鱼的生长情况.结果表明:经过三个月的养殖,在传统的养殖模式下,青田田鱼鱼苗平均末重为175.3 g/尾;本地鲤鱼平均末重为150.7 g/尾;在沟坑养殖模式下,青田田鱼的平均末重为230.3 g/尾,本地...     

福瑞鲤选育家系不同养殖阶段的生长差异分析

为了进一步观察最佳线性无偏预测(best linear unbiased prediction,BLUP)家系选育方法在福瑞鲤(Cyprinus carpio)继代选育中的潜力,该研究测量了继续选育第2代家系群体不同养殖阶段的体质量和形态性状。结果表明,生长快速家系群福瑞鲤早期(4月龄)生长速度较慢,到后期则生长加快,其体质量增长表现出明显的优势。在体型方面,随着养殖时间的延长,福瑞鲤各选育家系群的体厚/体长增加,体高/体长降低,逐渐呈现其体型修长的特征;同时2个越冬期的成活率均达到了94%以上。结果表明通过BLUP家系选育对福瑞鲤长期选育是可行的。在此基础上,通过主成分分析发现,福瑞鲤生长性状第一主成分是体质量;对不同生长时期的体质量进行相关性分析,发现9月龄、14月龄、21月龄鱼的体质量与24月龄的相关系数均达到极显著水平(P0.01),分别为0.851、0.897和0.957。因此,在福瑞鲤继续选育过程中,进行早期个体选择值得尝试。     

DNA polymorphism in the growth hormone gene and its association with weight in olive flounder Paralichthys olivaceus

ABSTRACT: Variation within the growth hormone gene and its association with growth trait in the olive flounder ( Paralichthys olivaceus ) was investigated. Based on Southern blot analysis and using various kinds of restriction endonucleases, it was demonstrated that the growth hormone gene exists as a single copy gene in the olive flounder. Polymorphisms of various lengths were also detected by Southern blot analysis, and by the subsequent digestion of the polymerase chain reaction (PCR) -amplified growth hormone gene fragment with Sau 3AI restriction enzyme. To study the possible association between variation in the growth hormone and weight, 60 progenies of the hatchery strain of three various sizes (large, medium and small weight) were selected and the entire genetic structure of the growth hormone gene was analysed. A total of 15 different genotypes was observed from the random association of six haplotypes. Significant heterogeneity of the growth hormone gene with haplotype and genotype frequencies was detected among the different-sized groups.     

Energy dynamics of subyearling Chinook salmon reveal the importance of piscivory to short‐term growth during early marine residence

Variation in prey quantity and quality can influence growth and survival of marine predators, including anadromous fish that migrate from freshwater systems. The objective of this study was to examine the energy dynamics of subyearling Chinook salmon (Oncorhynchus tshawytscha) following freshwater emigration. To address this objective, a population of Chinook salmon and their marine prey were repeatedly sampled from June to September over 2 years in coastal waters off Oregon and Washington. Subyearlings from the same population were also reared under laboratory conditions. Using a bioenergetics model evaluated in the laboratory, we found that growth rate variability in the field was associated more with differences in northern anchovy (Engraulis mordax) consumption and less with variation in diet energy density or ocean temperature. Highest growth rates (2.43–3.22% body weight/day) occurred in months when anchovy biomass peaked, and the timing of peak anchovy biomass varied by year. Our results support a general pattern among subyearling Chinook salmon occurring from Alaska to California that feeding rates contribute most to growth rate variability during early marine residence, although dominant prey types can differ seasonally, annually, or by ecosystem. In the northern California Current, faster growth appears to be associated with the availability of age‐0 anchovy. Identifying factors that influence the seasonal development of the prey field and regulate prey quantity and quality will improve understanding of salmon growth and survival during early marine residence.     

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。     

Effect of d-Lys6 salmon sGnRH alone and in combination with domperidone on the spawning of common carp during the late spawning season

The effect of [d-Lys6] salmon gonadotropin-releasing hormone alone (sGnRH) and in combination with domperidone, a dopamine antagonist, on the spawning of common carp, Cyprinus carpio, was investigated during the late spawning season (when mature fish stop spawning under natural conditions when males and females are put together). Fish were induced to spawn by [d-Lys6]-sGnRH salmon gonadotropin-releasing hormone analogue (sGnRH-X) only when injected in combination with domperidone at doses of 10 g kg-1 and 20 mg kg-1 body weight respectively. [d-Lys6]-sGnRH-X alone did not induce spawning at a dose as high as 100 g kg-1 body weight. Fish injected with domperidone alone did not spawn either. These results demonstrate that [d-Lys6]-sGnRH-X in combination with a dopamine antagonist is effective in inducing spawning during the late spawning season.