鲤鱼和草鱼基因文库的构建及其生长激素基因和肌动蛋白基因的筛选

用显微注射方法,把由小鼠重金属鳌合蛋白(MT)基因启动子驱动的人生长激素(hGH)基因导人鲤鱼等的受精卵内,由此发育而成的一部分鱼的基因组内携带有MThGH基因,这些鱼称之为"转基因鱼"1.       

鲤鱼生长激素基因在鲫鱼中的基因转移及PCR检测

鲫鱼群体内,个体之间的生长速度差别不大,遗传保守性较强,用常规育种方法很难获得生长速度快、个体差异较大的后代。通过基因转移技术,将外源基因如鱼的生长激素基因导入鱼的受精卵中,而获得转基因鱼是钱定向育种的一条有效途径。已有报道,在虹鳟鱼生长激素cDNA上游连结一个病毒启动子构建成表达质粒,由此获得的转基因鲤鱼比对照生长速度快20％。但是未见有将鲤鱼生长激素基因导入鲫鱼受精卵的报导。我们克隆了鲤鱼生长激素基因,并用它构建了含有病毒启动子（F.1,插入0．8Kb,位于XbaI和PstI之间)的表达质粒（Fig.2,命名为pCMV－TK－CGH）。用该质粒对鲫鱼受精卵进行注射,注射了1000粒卵,获得了转基因实验鲫鱼480尾。孵化率为48％。将其中100尾幼鱼于水簇箱中进行放养。选择其中个体较大的50尾进行PCR检测。其中8条鱼的基因组DNA有0．6Kb的PCR扩增产物（Fig.3),表明其上整合有外源的鲤鱼生长激素基因,其整合率为16％。其中最大的一条体重4．2g,体和6cm,比对照（0．7g,4cm）体重增加5倍,体长增加2cm(Fig.4)。我们构建的是高效表达质粒,其上含有巨细胞病毒增强子和人单纯疱疹病毒胸苷激酶启动子（属强启动子）,对鲤鱼生长激素基因有较强的启动作用。鲤鱼和鲫鱼在分类上属同科不同属,考虑到两者生长激素基因有较强的启动作用。鲤鱼和鲫鱼在分类上属同科不同属,考虑到两者生长激素基因的同源性,对转基因鲫鱼的PCR检测参照了加拿大学者Du所用引物的设计、使注射的外源基因与其本身的内源生长激素基因相区别。我们的转基因整合率（16％）与Zhang(10%)、Grass和Hackett等报道的接近。转基因鲫鱼生长速度增加的倍数（5倍）与Du等人的结果（2－6）接近。但是,这一生长速度快的优势能否遗传给后代,有待进一步观察。     

人生长激素基因在转基因鲤鱼体内的遗传

通过有性繁殖转移入生长激素基因鲤鱼(P₁),获得了转基因鲤鱼的子一代(F₁)和子二代(F₂);外源基因在转基因P1与普通鲤鱼杂交产生的F₁代和转基因F1代自交产生的F₂代鱼中的存在率分别为45．4％和66．7％,外源基因拷贝数在子代个体之间存在很大差异,从每细胞2拷贝到每细胞200拷贝不等;外源基因在转基因鱼子代中仍可表达具有生物功能的产物．人生长激素(h1GH),并能促进鱼的生长。     

鲤鱼生长激素基因在鲫鱼中的基因转移及PCR检测

鲫鱼群体内,个体之间的生长速度差别不大,遗传保守性较强,用常规育种方法很难获得生长速度快、个体差异较大的后代。通过基因转移技术,将外源基因如鱼的生长激素基因导入鱼的受精卵中,而获得转基因鱼是鱼类定向育种的一条有效途径。已有报道,在虹蹲鱼生长激素ｃＤＮＡ上游连结一个病毒启动子构建成表达质粒,由此获得的转基因鲤鱼比对照生长速度快２０％。但是未见有将鲤鱼生长激素基因导入鲫鱼受精卵的报导。我们克隆了鲤鱼生长激素基因,并用它构建了含有病毒启动子（Ｆｉｇ．１,插入片段为０．８Ｋｂ,位于ＸｂａＩ和ＰｓｔＩ之间）的表达质粒（Ｆｉｇ．２,命名为ｐＣＭＶ－ＴＫ－ＣＧＨ）。用该质粒对鲫鱼受精卵进行注射,共注射了１０００粒卵,获得了转基因实验鲫鱼４８０尾,孵化率为４８％。将其中１００尾幼鱼于水簇箱中进行放养。选择其中个体较大的５０尾进行ＰＣＲ（检测。其中８条鱼的基因组ＤＮＡ有０．６Ｋｂ的ＰＣＲ扩增产物（Ｆｉｇ．３）,表明其上整合有外源的鲤鱼生长激素基因,其整合率为１６％。其中最大的一条体重４．２ｇ,体长６ｃｍ,比对照（０．７ｇ,４ｃｍ）体重增加５倍,体长增加２ｃｍ（Ｆｉｇ．４）。我们构建的是高效表达质粒,其上含有巨细胞病毒增强子和     

两个人生长激素基因在SV40病毒重组体感染的猴细胞中的表达

我们已经建造了带有两个不同的人生长激素(hGH)基因的SV40重组体。用这些重组体感染的猴肾细胞能合成、加工并分泌人生长激素。有着与克隆的人生长激素互补DNA(eDNA)同样编码序列的基因1,共产物从几个标准来看,与垂体hGH没有什么区别。预定编码一个变异蛋白质的基因2,其产物比垂体hGH的免疫反应活性要小,但能有效地与hGH细胞表面受体结合。这些结果表明,基因2有可能表达而产生我们以前未辨别的hGH形式。这些结果显示了在真核细胞中,用基因转移的方法产生成熟的激素是可能的。这些结果也证明了SV40—猴细胞系统可以用于生产和鉴定动物细胞分泌的蛋白质。     

转移人生长激素基因和注射人生长激素对促进银鲫生长的研究

本研究将人生长激素基因重组 DNA 导入银鲫受精卵,或用人生长激素对60日龄银鲫连续6周腹腔注射,均观察到了受体鱼的快速生长效应,转移人生长激素基因鱼在60日龄时的平均体重比对照鱼平均体重增加82%,相应体长平均值比对照鱼增加19.7%;在125日龄时,转基因组平均体重比对照组增加17.7%,相应体长平均值比对照增加3.5%。定期注射人生长激素,在6周的注射实验过程中,实验鱼的体重,体长都与对照组相近或略有减小。但是,在水泥池中饲养一个月后,注射10/μg/g 体重的实验组鱼平均体重比相应对照组鱼的平均体重增加32%;相应体长平均值增加11%。     

快速生长转基因鲤鱼可望在中国实现商品化

由中国科学院院士、中国科学院水生生物所朱作言研究员主持的“快速生长转基因鲤鱼的中试研究”已通过成果鉴定。中国科学院李振声院士、中国工程院刘筠院士和林浩然院士等专家鉴定认为,该项研究建立了快速生长转基因鲤鱼的高效、安全养殖模式;对转“全鱼”生长激素基因鱼食品消费安全进行了严密的科学实验,证实了转“全鱼”生长激素基因鱼的食品消费安全性,为转“全鱼”生长激素基因鲤鱼的大规模商品化生产提供了科学依据,研究成果居国际领先水平。 朱作言院士主持的该项研究,筛选获得了快速生长的转“全鱼”生长激素基因黄河鲤鱼核心群200尾,其生长速度比对照鱼快140％以上。试验证明：转基因鱼怀卵量略低于普通黄河鲤鱼,受精率和孵化率与对照鱼无显著差异,转“全鱼”生长激素基因黄河鲤鱼具备大规模苗种繁育生产能力;转“全鱼”生长激素基因鲤鱼F1代的大规模养殖不仅增产,而且可降低养殖成本,并为转基因鱼的进一步选育提供材料;转“全鱼”生长激素基因三倍体鱼平均体重增长速度比对照鱼提高15％,铒料利用效率提高11．1％,由此建立了转“全鱼”生长激素基因高效、安全的养殖模式。试验还证明：摄食转“全鱼”生长激素基因鱼对小鼠的生长、脏器发育、血液生理生化指标、繁殖能力及其后代的生长发育均无影响。为此,专家建议,尽快推广养殖转基因三倍体鲤鱼,在我国建立世界首例转基因动物品种商品化生产的范例。杨淑培     

在细菌中合成人生长激素

控制着人生长激素（(hGH),催乳激素以及 绒毛膜生长激素的各个基因有着共同的起源, 因此,这组基因是研究具有亲缘关系的某些基 因的结构、进化和分化调节的极好模型。hGH 又是治疗垂体衰退性矮小症的特效药物,在治 疗其它失调性疾病上,疗效也很显著。但是,由 于它只能从死尸的垂体中提取,所以远远不能 满足需要。如果能在细菌中合成hGH,对于开 展理论研究和医疗实践都具有重要意义。     

生长激素

19世纪末人们在临床工作中已发现生长发育与垂体有关。1912年Ashner发现单纯切除垂体前叶导致生长障碍。1921年Evans等证实牛垂体提取液对大鼠有促进生长作用。1943年Evans建立了生长激素GH的生物学测定方法即胫骨测定法。1944年李卓浩和伊万斯提纯了牛GH,确定了垂体GH的存在。1956年李卓浩等又提纯了人生长激素(hGH),发现GH有高度种属特异性,并使hGH应用于临床治疗有了可能。1962年Hunter等建立了hGH的放射免疫测定法,从而建立了各种hGH功能试验,使hGH功能紊乱疾病包括垂体侏儒的诊断水平显著提高。1971年已能     

转基因鱼

本文综述了转基因鱼研究的进展,并介绍了目的基因的选择及其导入的主要方法。此外,还对转基因鱼研究中尚存的一些问题作了说明。 自从八十年代中期开始鱼类基因转移研究以来,进展非常迅速。1985年Zhu等成功地将带有小鼠重金属结合蛋白(mMT)启动子的人生长激素(hGH)基因导入鲫鱼受精卵,并获得生长速度快的子代鱼。随后,世界一些国家开展了鱼类基因转移的研究,并取得很大成就。本文拟对转基因鱼研究现状及尚存的问题作一概述,供研究者参考。     

Absorption of bioactive human growth hormone after oral administration in the common carp (Cyprinus carpio) and its enhancement by deoxycholate

Summary Recombinant human growth hormone was administered orally to carp and serum levels of absorbed bioactive hormone were investigated using a highly sensitive Nb2 rat lymphoma cell bioassay and radioimmumoassay. Serum levels of bioactive hGH reached maximum values 30 min after oral intubation and then gradually decreased. Co-administration of the hormone with deoxycholate to fasted carp resulted in up to a 1000-fold increase in absorption compared to aqueous solutions of the hormone, but had no effect on the kinetics of the absorption process. Absorption of the hormone in starved fish was significantly greater than in fed fish. A linear dose-response relationship was observed for hGH in starved fish and the level of absorption in fed fish was influenced by the time interval from the last meal. The ratio of bioactive to immunoactive hGH in fasted fish indicated little loss of bioactivity and also that deoxycholate may be protective against hGH degradation. The present study demonstrates for the first time that biologically active hGH is absorbed in the common carp after oral intubation. Furthermore, the use of a biological detergent dramatically increased the extent of hGH absorption. Additional studies are required to establish the approapriate conditions (diet composition, feeding level, and frequency, etc.) in which polypeptide hormones could be introduced orally to fish.Abbreviations hGH human growth hormone - HRP horseradish peroxidase - RIA radioimmunoassay     

全鱼基因的构建及其在鲫鱼体内的整合与转录

利用PCR技术删除大麻哈鱼生长激素基因的启动序列,通过基因重组构建出全鱼基因（鲤鱼MT启动子－大麻哈鱼生长激素基因）;以融合全鱼基因为外源基因,通过显微注射方法将其线性片段导入鲫鱼受精卵内,研究其整合与转录效率。结果表明,全鱼基因在鲫鱼基因组中的整合率为36．4％（16／44）,对转基因阳性鱼的RNA样本进行Northern印迹杂交检测,转录率为25％（1／4）。因此,该全鱼基因可以作为转基因鱼研究和应用的外源基因。     

从ATPase8-6基因研究杂交多倍体鱼线粒体母性遗传

异源四倍体鲫鲤是世界上首例人工培育的两性可育并形成群体的且能自然繁殖的四倍体鱼。本文采用质粒克隆测序法测定了红鲫、异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤的ATPase8和ATPase6基因全序列 ,结合鲤鱼、日本白鲫和斑马鱼的同源序列 ,对不同倍性水平鲤科鱼类的ATPase8和ATPase6基因进行了比较 ,分析了碱基组成、变异情况以及核苷酸和氨基酸序列差异。红鲫、鲤鱼、异源四倍体鲫鲤、日本白鲫、三倍体湘云鲫和三倍体湘云鲤之间的序列差异为 0 0 % - 1 3 4 % ,它们与外群斑马鱼之间的序列差异为 2 7 9% -31 0 %。用MEGA软件中的MP法、ME法、NJ法和UPGMA法构建分子系统树 ,得到了相似的拓扑结构。结果分析表明 ,人工杂交多倍体异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤在线粒体ATPase8和ATPase6基因上具有严格的母性遗传特征。值得注意的是 ,异源四倍体鲫鲤经过 1 1代的繁育后 ,与其原始母本红鲫仍然保持了非常高的同源性 ,说明了新的异源四倍体基因库在线粒体ATPase8和ATPase6基因上拥有稳定的遗传特性。对不同倍性鲤科鱼类线粒体ATPase8和ATPase6基因的研究表明 ,ATPase8和ATPase6基因是杂交鱼后代遗传变异研究的一个很好的分子标记     

长江中游典型草型湖泊与藻型湖泊鲫种群结构和生长比较(英文)

浅水湖泊牛山湖和月湖均位于长江中游武汉市境内,前者水质清澈、沉水植物茂盛,属典型的草型湖泊;后者长期受生活污水和工业废水排放的影响,水质恶劣、沉水植物稀少,属典型的藻型湖泊。文章选择这两个湖泊的共同优势鱼类——鲫(Carassius auratus)作为研究对象,通过比较两类不同湖泊环境条件下鲫种群结构和生长上的差异,评估人类活动引起的水环境改变对鱼类种群生存状况的影响。2006年春季(3—5月),于牛山湖和月湖各采集鲫种群样本713尾和641尾,研究结果表明:(1)牛山湖和月湖鲫种群的全长频数分布经kolmogorov-Smirnov检验均符合正态分布,但前者的分布范围(63—271 mm)大于后者(72—191 mm),前者的全长峰值(190 mm)亦大于后者(110 mm);(2)牛山湖鲫种群的年龄组成(6个年龄组)较月湖(仅3个年龄组)完整,但月湖各龄鲫的全长均显著大于牛山湖(P<0.05);(3)两个湖泊性成熟鲫的雌、雄鱼比均随全长的增长而增加,即小个体性成熟鲫以雄鱼为主,大个体性成熟鲫以雌鱼为主,该现象在月湖更为明显;(4)牛山湖和月湖鲫全长–体重回归关系(W=a Lb)分别为W=0.00001 L3.060(R2=0.989)和W=0.000006 L3.246(R2=0.971),协方差分析表明两个湖泊鲫种群的b值差异极显著(P<0.01);另一方面,月湖鲫种群各全长组雌、雄鱼的肥满度均显著大于牛山湖(P<0.05)。作者认为影响牛山湖和月湖鲫生长和种群结构的主要因素是两个湖泊饵料组成和捕捞压力等方面的差异。     

Gene transfer in common carp (Cyprinus carpio L.) by microinjection into the germinal disc

We report here on the microinjection of the human growth hormone gene (hGH) into the germinal disc of common carp one-cell embryos. For microinjection, a linear 4.1 kb EcoRi-DNA fragment containing the mouse metallothionein-I promoter (mMT-I), fused to a structural gene coding for the hGH, was used. Of the 14 injected embryos, 9 developed to an age of 60 d. Of these nine fish, two were found to be transgenic and had integrated two to four copies per cell of the hGH construct.     

Expression of a Novel Piscine Growth Hormone Gene Results in Growth Enhancement in Transgenic Tilapia (Oreochromis Niloticus)

Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced following egg injection with gene constructs carrying growth hormone coding sequences of fish origin. Using a construct in which an ocean pout antifreeze promoter drives a chinook salmon growth hormone gene, dramatic growth enhancement has been demonstrated, in which the mean weight of the 7 month old G2 transgenic fish is more than three fold that of their non transgenic siblings. Somewhat surprisingly G1 fish transgenic for a construct consisting of a sockeye salmon metallothionein promoter spliced to a sockeye salmon growth hormone gene exhibited no growth enhancement, although salmon transgenic for this construct do show greatly enhanced growth. The growth enhanced transgenic lines were also strongly positive in a radio-immuno assay for the specific hormone in their serum, whereas the non growth enhanced lines were negative. Attempts to induce expression from the metallo thionein promoter by exposing fish to increased levels of zinc were also unsuccessful.Homozygous transgenic fish have been produced from the ocean pout antifreeze/chinook salmon GH construct and preliminary trials suggest that their growth performance is similar to that of the hemizygous transgenics. No abnormalities were apparent in the growth enhanced fish, although minor changes to skull shape and reduced fertility were noted in some fish. There is also preliminary evidence for improved food conversion ratios when growth enhanced transgenic tilapia are compared to their non-transgenic siblings.The long term objective of this study is to produce lines of tilapia which are both growth enhanced and sterile, so offering improved strains of this important food fish for aquaculture.     

A laboratory study of the effects of shelter availability and invasive crayfish on the growth of native stream fish

1. The alteration of stream habitats by urbanisation reduces the availability of shelter. Reduced shelter availability may increase both predation risks and metabolic costs, negatively affecting the growth performance of stream fish. Although urbanised streams often allow the establishment of invasive species, the additive or interactive effects of shelter availability and invasive species are rarely explored. The invasive red swamp crayfish (Procambarus clarkii) is a strong shelter competitor and predator in streams. 2. We conducted a stream‐channel experiment to investigate how shelter availability and this invasive crayfish species affect the growth performance (measured as the loss in body mass under starved conditions) of two native benthic species, Japanese crucian carp (Carassius auratus complex) and an endangered cobitid fish (Lefua echigonia). 3. We found that both the absence of shelter and the presence of crayfish increased body mass loss in the crucian carp. However, the growth performance of the crucian carp was improved by the presence of shelter except in the presence of crayfish, in which case the shelter available did not ameliorate the negative effect of the crayfish on growth performance (i.e. an interactive effect). This result suggests that crayfish may affect the growth performance of the crucian carp through chemical cues as well as through shelter occupancy. 4. In contrast, shelter availability is the primary factor affecting the body mass loss of the cobitid fish. However, the growth performance of these fish was unaffected by the presence of crayfish. The change in the growth performance of the cobitid fish in response to the absence of shelter was greater than that of the crucian carp. This finding suggests that the cobitid fish may be more vulnerable than the crucian carp to a reduction in shelter availability. 5. Our study demonstrates that reduced shelter availability and/or crayfish invasion can have significant, indirect negative effects on the growth performance of native fish, but whether those effects are interactive might vary depending on the species. Our findings have clear implications for stream restoration and habitat assessment.     

Comparative and Evolutionary Analysis of the Cytochrome b Sequences in Cyprinids with Different Ploidy Levels Derived from Crosses

The mitochondrial cyt b genes in the allotetraploid and triploid crucian carp as well as triploid common carp were isolated and completely sequenced. Their DNA sequences were compared with those derived from the cyt b genes of the red crucian carp, Japanese crucian carp, and common carp with MEGA 1.0 software. Phylogenetic analysis revealed the sister relationships between allotetraploid and diploid red crucian carp, between the triploid crucian carp and diploid Japanese crucian carp, and between triploid common carp and diploid common carp. Our results indicated the cyt b genes in the allotetraploid, triploid crucian carp, and triploid common carp were maternally inherited. Through maternal inheritance, the cyt b gene in the F11 tetraploid displayed extremely high similarity to that in the female parent red crucian carp after 11 generations (from F1 to F11 hybrids). Since the establishment of the new tetraploid stocks has great significance in analyzing evolutionary theory of vertebrate and in improving aquaculture industry, analysis of the cyt b gene and the elucidation of the variation of the cyt b gene DNA in different cyprinids prove that cyt b is a useful genetic marker to monitor the variations in the progeny of the crosses.