转大麻哈鱼生长激素基因鲫鱼研究

通过显微注射方法得到了一批转大麻哈鱼生长激素基因的实验鲫鱼。斑点杂交和ＳｏｕｔｈｅｒｎＢｌｏｔ杂交证实部分实验鱼的基因组中已整合了外源生长激素基因,并有一尾鱼有比较明显的生长速度快的特征。     

大麻哈鱼生长激素基因Ⅱ的分子克隆和序列分析

以λ噬菌体ＥＭＢＬ－３为基因载体,构建大麻哈鱼（Ｏｎｃｏｒｈｙｎｃｈｕｓ　ｋｅｔａ）基因组文库,并从中分离出含全长的生长激素（ｃｓＧＨ）基因的克隆。本研究首次报道了对ｃｓＧＨ全长核苷酸序列分析的结果。发现ｃｓＧＨ基因由６个外显子和５个内含子组成,长度为３３６１碱基对（ｂｐ）。由该序列可推导出含２１０个氨基酸的多肽,其中存在２２个疏水氨基酸残基的信号肽。本研究所分析的ｃｓＧＨ的外显子和内含子,其排列方式与其他鲑类（Ｓａｌｍｏｎｉｄｓ）和罗非鱼（Ｔｉｌａｐｉａ）是相似的,而与鲤科鱼类（Ｃａｒｐｓ）不同,后者外显子和内含子的排列方式与哺乳类和鸟类相似。将所测定的 ｃｓＧＨ基因的编码区与已发表的 ｃｓＧＨⅡ和ｃｓＧＨ Ⅱ两种 ｃＤＮＡ序列进行比较,证明本研究所克隆的 ｃｓＧＨ基因应属于 ｃｓＧＨ Ⅱ,因它们的编码序列 １００％同源。     

转青鱼生长激素基因异源四倍体鲫鲤

生态安全性是转基因鱼走向市场的瓶颈,通过转基因四倍体鱼同转基因二倍体鱼杂交获得不育的转基因三倍体鱼是解决该问题的有效途径之一.本研究构建了青鱼β-actin基因启动子和青鱼生长激素（GH）基因精确连接的＂全鱼＂基因pbcAbcGHc;并采用显微注射法将pbcAbcGHc导入异源四倍体鲫鲤受精卵.对照养殖结果表明,150日龄的转基因异源四倍体鲫鲤原代（P0）的体重及体长明显大于对照组.选择60尾P0代转基因异源四倍体鲫鲤,采用PCR方法检测出外源青鱼GH基因在P0代转基因四倍体尾鳍基因组DNA中的整合率为90%;对20尾雄性P0代转基因四倍体精液样本的PCR检测发现,13个样本具有外源青鱼GH基因的整合.在一尾生长速度显著的P0代转基因四倍体鲫鲤的肌肉、肝脏、肾脏和卵巢组织中可检测到外源青鱼GH基因的转录.本研究成功获得了具有明显生长优势的P0代转青鱼GH基因异源四倍体鲫鲤,为建立转青鱼GH基因异源四倍体鲫鲤纯系和研制不育的转基因三倍体鱼奠定了基础.     

转大麻哈鱼生长激素基因鲤表型性状与体质量的相关性及通径分析

随机选取二龄转大麻哈鱼生长激素基因鲤和对照鲤各30尾,运用相关分析和通径分析的方法研究全长、体长、体高、尾柄高、尾柄长、头长、吻长、眼径、眼间距、体厚10个表型性状对体质量的影响程度,以此确定影响二龄转基因鲤和对照鲤体质量的主要表型参数.相关分析结果表明:转基因鲤和对照鲤的大部分表型性状与体质量间的相关系数均达到极显著水平(P<0.01).通径分析结果显示:体长和体高可以作为预测转基因鲤体质量的主要表型参数,转基因鲤体长对体质量的通径系数为0.572,体高对体质量的通径系数为0.415,体长和体高直接决定体质量;而体厚和头长可以作为预测对照鲤体质量的主要表型参数,对照鲤体厚对体质量的通径系数为0.610,头长对体质量的通径系数为0.377,体厚和头长对体质量具有决定作用.     

建鲤生长激素受体基因分离、转录子多态性以及组织表达特性

使用PCR、RT-PCR和RACE方法分离克隆了建鲤基因组内的4个jlGHRs基因,同源性分析和系统树表明它们两两分属于鱼类GHR1和GHR2,命名为jlGHR1a、jlGHR1b;jlGHR2a、jlGHR2b。jlGHR1s和jlGHR2s的两个旁系同源基因间氨基酸差异分别为5%和11%,但功能保守区FGVFS基序、Box1、Box2基本一致,jlGHR1s和jlGHR2s氨基酸差异为41%。jlGHRs和斑马鱼GHRs基因结构相同,在阅读框内存在7个内含子,两旁系同源基因间内含子长度或序列存在差异。jlGHR1s、jlGHR2s与不同鱼类GHR1、GHR2同源性高低与传统分类地位一致。实验过程中发现建鲤肝脏存在jlGHRs的多种转录子,包括丢失了外显子4的jlGHR1b’、保留了内含子3的jlGHR2a’、丢失了部分外显子8的jlGHR2as。实时定量RT-PCR组织表达结果显示4个jlGHRs在脑、肝、心、头肾、肾、肠、脾、肌肉各组织中均有表达,但表达量差异明显,其中肌肉组织中4个基因表达量均最高,脑中4个基因的表达水平相当,其余各组织中jlGHR2b的表达量均最高。从多转录子和较低表达量推测jlGHR2a所受的选择压力低于jlGHR2b。鲤鱼基因组内分离到GHR1、GHR2的两个旁系同源基因在功能基因方面证实了鲤鱼体内存在两套基因,表明鲤鱼是研究同源基因变异分化的好材料,也为今后正确查找jlGHRs基因上的SNP位点奠定了基础。     

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。     

转抗虫基因植物生态安全性研究进展

转抗虫基因植物如Bt棉花等已在美国、中国和澳大利亚等国家大规模商业化种植 ,有关转抗虫基因植物潜在的生态风险已引起广泛的关注。该文综述了转抗虫基因植物研究应用现状与安全性研究进展。主要内容包括 :转抗虫基因植物的种类及其对靶标害虫的抗性 ,对非靶标害虫和天敌发生的影响 ,对农田生态系统生物多样性的影响 ,靶标昆虫的抗性治理及转抗虫基因植物的基因漂移等     

中华鳖转“全鱼”生长激素基因研究

自首例快速生长转基因鱼在我围诞生以来[1],世界上很多试验室相继开展了转基因鱼研究,以期获得快速生长[2,3]、抗逆[4,5]的养殖新品系.与传统的杂交选育方法相比,转基因育种方法摆脱了物种间遗传壁垒的桎梏,基于对目的基因功能的认识和把握,定向培育优良养殖品种,大幅缩短育种周期.     

转Bt基因玉米的生态安全性研究进展

随着转基因作物的应用和推广 ,转 Bt基因作物释放后对生态环境及其它方面产生的潜在影响越来越受到重视。分别从生物活性杀虫晶体蛋白在土壤中的残留特性、杀虫晶体蛋白对土壤中非目标生物的影响、转 Bt基因玉米植株体成分的变化、转Bt基因玉米花粉中杀虫晶体蛋白的表达特性及其在田间和马力筋叶片上的散积状况、花粉中表达的杀虫晶体蛋白对君主斑蝶的毒性、君主斑蝶幼虫暴露在 Bt花粉中的概率及综合风险评价估算等方面对转 Bt基因玉米产生的杀虫晶体蛋白与土壤生态环境的相互作用、花粉对非目标生物影响的研究现状进行了综述。通过对转 Bt基因作物生态安全性的科学评价和广泛宣传 ,以确保生物技术的健康发展。     

外源猪生长激素基因在金鱼体内的整合与表达研究

自1982年Palmiter^[1]等人给小鼠受精卵雄原核注射大鼠生长激素基因培养成功“超级”鼠以来,由于人们认识到转基因技术导致动植物品种定向改良以及利用转基因动物作为生物反应器,大量合成和分泌贵重而安全的基因工程产品,因此转基因动物技术得到了迅速发展,相继开展了兔、鱼、猪、鸡、牛、羊等动物的转基因研究,并取得了一定的结果^[2,3,4]。但是在上述具有经济价值的高等脊椎动物中从事转基因研究,成本高、操作困难,而金鱼属于低等脊椎动物．具有产卵多、易获得同步卵、可控制体外受精和体外发育等特点,因而成为转基因动物研究的方便材料。生长激素是动物脑下垂体前叶分泌的单链多肽⑸,生理功能是促进碳水化合物代谢及核酸、蛋白质合成,整体效应表现为动物生长。本文以金鱼为实验材料,探讨猪生长激素基因导入其受精卵后整合、表达以及生物效应等问题,为进一步研究外源基因在高等动物内整合和表达调控机理提供参考。     

Growth and energy budget of F2'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F₂‘all‐fish’ growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29·2° C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I_E), and the proportion of I_E utilized for heat production (H_E) were significantly higher in the transgenics than in the controls. The proportion of I_E directed to waste products [faecal energy (F_E) and excretory energy loss (Z_E+U_E) where Z_E is through the gills and U_E through the kidney], and the proportion of metabolizable energy (M_E) for recovered energy (R_E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I_E= 19·3 F_E+ 6·0 (Z_E+U_E) + 45·2 H_E+ 29·5 R_E or 100 M_E= 60·5 H_E+ 39·5 R_E. The average energy budget equation of the controls was: 100 I_E= 25·2 F_E+ 7·4 (Z_E+U_E) + 35·5 H_E+ 31·9 R_E or 100 M_E= 52·7 H_E+ 47·3 R_E. These findings indicate that the high growth rate of ‘all‐fish’ transgenic common carp relative to their non‐transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.     

Fast-growing transgenic common carp mounting compensatory growth

Compensatory growth is a phase of accelerated growth apparent when favourable conditions are restored after a period of growth depression. To investigate if F₂ common 'all-fish' growth hormone gene transgenic common carp ( Cyprinus carpio ) could mount compensatory growth, a 9 week study at 29° C was performed. The control group was fed to satiation twice a day throughout the experiment. The other two groups were deprived of feed for 1 or 2 weeks, respectively, and then fed to satiation during the re-feeding period. At the end of the experiment, the live masses of fish in the deprived groups were still significantly lower than those of the controls. During the re-feeding period, size-adjusted mean specific growth rates and mean feed intakes were significantly higher in the deprived fish than in the controls, indicating a partial compensatory growth response in these fish. No significant differences were found in food conversion efficiency between the deprived and control fish during re-feeding, suggesting that hyperphagia was the mechanism responsible for increased growth rates. The proximate composition of the deprived fish at the end of the experiment was similar to that of the control fish. This study is, to our knowledge, the first to report that fast-growing transgenic fish can achieve partial compensation of growth following starvation.     

Preliminary studies on the genetic variability of six Hungarian common carp strains using microsatellite DNA markers

Genetic variation in six Hungarian common carp (Cyprinus carpio L.) strains was evaluated using 12 microsatellite loci. The domesticated (Tatai, Biharugrai and Szarvasi) strains were derived from fish farms. Two of wild strains (Tiszai and Dunai) were sampled from brood stocks maintained at fish farms for breeding, and Kis-Balatoni wild carp were sampled from the Small Balaton Lake. Pairwise Fst-values (0.013–0.161) were highly significant (p<0.0001), demonstrating differentiation among strains. The mean number of alleles ranged between 3.9 and 8.2. Overall mean observed heterozygosity was lower (0.557) than the mean expected heterozygosity (0.700). By strain, the only exception to this trend was the Dunai (Danubian), which showed higher mean observed heterozygosity (0.764) than expected (0.602). For five loci the Dunai strain showed extremely high levels of heterozygosity (1.00). Two wild strains exhibited a number of loci (Tiszai, 4; Dunai, 6) that were not in Hardy–Weinberg equilibrium. A relatively high number of private alleles overall (n=26), as well as differences in allele frequencies supported our ability to assign most individual fish (over 90%) to each strain.     

A one-year investigation of the relationship between serum GH levels and the growth of F4 transgenic and non-transgenic common carp Cyprinus carpio

It has been demonstrated that growth hormone (GH) transgenic fish often posses a trait for fast growth. Here, we investigated the growth of F₄'all-fish' GH transgenic carp Cyprinus carpio and their serum GH levels for a year. The results showed that F₄ all-fish GH transgenic carp were significantly larger in body mass ( c . two-fold, P < 0·001) and body length ( c . 1·3 fold, P < 0·001), compared with the non-transgenic group. The discrepancy of serum GH levels between the transgenic carp group and control group is 54 fold, when the water temperature was 12–34° C. When the water temperature decreased to 3·5° C in January, the discrepancy was 256 fold. The serum GH level of the transgenic group was relatively constant, while that of control varied greatly based on month and water temperature. The changes of growth rates between the transgenic group and the control group were similar for a year. Taken together, the results indicated that F₄ all-fish GH transgenic carp had not only higher and constant serum GH levels but also a significant fast-growing effect, compared with the control. To our knowledge, this is the first report on a one-year investigation of growth trait and serum growth hormone level in F₄ all-fish GH transgenic carp.     

Detection of allelic variation within the growth hormone gene in common bream using heteroduplex analysis*

A novel method, heteroduplex analysis, was applied for detecting variation in an amplified growth hormone (GH) gene segment of common bream Abramis brama. Two-allele polymorphism was detected within a 262 bp restriction fragment. The frequency of the variant haplotype in the Main River was 0–50 and in the Danube River 0–32, but the difference was Statistically not significant.     

Co-injection strategy improves integration efficiency of a growth hormone gene construct,resulting in lines of transgenic tilapia (Oreochromis niloticus) expressing an exogenous growth hormone gene

A co-injection strategy was employed to improve the efficiency of integration of a poorly integrating but commercially important growth hormone gene construct in tilapia. Its co-injection with a reporter gene construct of higher integration efficiency yielded a threefold increase in the integration efficiency of the growth hormone gene construct. In addition, out of 13 transgenic founder tilapia generated, three transmitted the transgenes to G1 and G2 progeny with expected Mendelian inheritance ratios in the G2 generation. We also observed expression of both constructs in a number of founder and G1/G2 individuals. Evidence is provided for the co-ligation of the two constructs and we suggest that this accounts for the increased integration efficiency of growth hormone gene construct and its successful transmission and expression, thus generating lines of novel growth hormone expressing tilapia     

Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T, injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about ?35–40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly ?25–32 and ?29–41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by ?72–81 and ?51–70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels. © 1995 Wiley-Liss, Inc.     

榕江香猪生长激素基因的鉴定及功能分析

生长激素是调节动物生长的主要激素.本研究应用聚合酶链式反应技术从榕江香猪的基因组文库中分离出1.903kb生长激素基因.克隆的生长激素基因由五个外显子和四个内含子组成.榕江香猪生长激素基因的碱基序列与已知四个国外猪种和9个中国地方猪种之间的同源性为97%～99%,其间的差异主要集中在内含子2和4.通过限制性内切酶（DdeI,NarI,BsmNI）分析,鉴定出榕江香猪生长激素基因的五个多态性位点,分别位于5＇-侧翼区274（T/C）位点,外显子2的622（G/A）和631（G/A）位点,内含子2中的841（T/C）以及外显子4中的1 358（A/G）位点.同时,1 358（A/G）位的碱基改变导致榕江香猪生长激素成熟肽第108位异亮氨酸替换,三维结构分析表明,异亮氨酸的存在可能导致生长激素与受体间亲合力降低.     

猪生长激素研究进展

猪生长激素是由猪脑垂体前叶嗜酸性细胞分泌的一种单一肽链的蛋白质激素。本文就猪生长激素基因的结构特点,多肽性研究、基因表达及猪生长激素的结构特点、生理功能、分泌规律及与其抗体的相互作用等方面进行了综述。     

Elevated ability to compete for limited food resources by 'all-fish' growth hormone transgenic common carp Cyprinus carpio

Food consumption, number of movements and feeding hierarchy of juvenile transgenic common carp Cyprinus carpio and their size-matched non-transgenic conspecifics were measured under conditions of limited food supply. Transgenic fish exhibited 73·3% more movements as well as a higher feeding order, and consumed 1·86 times as many food pellets as their non-transgenic counterparts. After the 10 day experiment, transgenic C. carpio had still not realized their higher growth potential, which may be partly explained by the higher frequency of movements of transgenics and the 'sneaky' feeding strategy used by the non-transgenics. The results indicate that these transgenic fish possess an elevated ability to compete for limited food resources, which could be advantageous after an escape into the wild. It may be that other factors in the natural environment ( i.e. predation risk and food distribution), however, would offset this advantage. Thus, these results need to be assessed with caution.