两种不同终止子在转基因鲤鱼中的促生长效应

转基因构建体中启动子的选择会直接影响转植基因的活性, 近年来有研究表明转基因构建体中终止子的选择会一定程度地影响转植基因的活性。为了更好地筛选转基因构建体和培育快速生长的转“全鱼”生长激素(Growth hormone, GH)基因鱼, 文章用鲤鱼β-actin基因终止子和生长激素基因终止子分别构建了转基因构建体, 显微注射得到转“全鱼”GH基因鱼P0代养殖群体, 比较两种不同终止子构建体的活性。统计分析发现, 生长激素基因终止子构建体的养殖群体的体重频率呈正态分布且平均体重显著高于β-actin基因终止子构建体的养殖群体, β-actin基因终止子构建体的养殖群体的体重频率呈右倾趋势的非正态分布。值得一提的是在混合养殖组中得到一条生长最为快速的鲤鱼证实为转基因阳性且为生长激素基因终止子构建体的转基因鲤鱼。该结果表明转“全鱼”生长激素基因鲤鱼可快速生长, 并能将转植基因向下代遗传。实验结果提示生长激素基因终止子构建体比β-actin基因终止子构建体表现的促生长活性要强。     

转CpTI-Bt基因棉和转Bt基因棉对棉铃虫幼虫存活、生长及营养利用的影响

2000年7月中旬和8月中旬, 分别测定了采自田间的转CpTI-Bt基因双价抗虫棉（SGK321, 以下简称CpTI-Bt棉）和转Bt基因抗虫棉（中30,以下简称Bt棉）对棉铃虫Helicoverpa armigera幼虫存活、生长的影响。结果表明：7月中旬两种转基因抗虫棉抗虫效果均较好,尤其是CpTI-Bt棉棉叶和花瓣对4龄幼虫3天内致死率为92%以上;8月中旬两种转基因棉的抗虫活性均明显降低,且Bt棉的杀虫活性显著低于CpTI-Bt棉,其幼虫死亡率与对照受体棉中16的死亡率之间无显著差异,仅显著抑制了幼虫的生长;石远321(SGK321受体品系)的花瓣具有一定的抗虫活性,可显著降低取食幼虫的体重,甚至造成部分幼虫死亡; CpTI-Bt棉中,花瓣和棉叶的抗虫性明显高于蕾和铃心。对5龄幼虫取食棉铃1日后的营养指标测定结果显示： 两种转基因抗虫棉处理的幼虫相对生长率和相对取食量均显著低于石远321,但两者之间无显著差异; CpTI-Bt棉处理的幼虫近似消化率显著低于石远321和Bt棉,但其食物利用率显著高于石远321和Bt棉。     

Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats

The production of valuable pharmaceutical proteins using transgenic animals as bioreactors has become one of the goals of biotechnology. However, the efficiency of producing transgenic animals by means of pronuclear microinjection is low. This may be attributed in part to the low integration rate of foreign DNA. Therefore, a large number of recipients are required to produce transgenic animals. We recently developed a transgenic procedure that combined the techniques of goat oocyte in vitro maturation (IVM), in vitro fertilization (IVF), microinjection, preimplantation selection of the transgenic embryos with nested PCR and transferring the transgenic embryos into the recipient goat uterus to produce transgenic goats. Thirty-seven transgenic embryos determined by nested PCR were transferred to thirty-two recipient goats. In the end, four live-born kids were produced. As predicted, all the live kids were transgenic as identified by PCR as well as Southern blot hybridization, The integration rate was 100% (4/4) which was completely in accordance with the results of embryo preimplantation detection. The results showed a significant decrease in the number of recipients required as only 8 recipients (32/4) were needed to obtain one live transgenic goat. We suggest that the transgenic system described herein may provide an improved way to efficiently produce transgenic goats on a large scale.     

转基因植物根系分泌物对土壤微生态的影响

随着转基因植物商品化进程的加快,对其进行生态风险性评价日益引起学者的重视。诸如转基因逃逸到其它亲缘物种中、产生超级杂草和病毒、昆虫产生耐受性及生物多样性遭受破坏等问题已在部分转基因作物中显现。本文综述了转基因植物中根系分泌物对土壤微生态的影响。     

转基因植物环境监测进展

近20年来,转基因植物的商业化应用规模越来越大,而转基因生物安全问题依然是转基因植物产业进一步发展的最主要制约因素。转基因植物在商业化应用之前虽然预先进行了风险评估,但是,包括环境监测在内的风险管理措施是确保转基因植物安全应用的必要手段。在转基因作物大规模应用近20年之后,其在靶标生物抗性、对生物多样性的影响、基因漂移、在生态系统中的长期存留等方面产生的环境风险已经渐渐显现出来,表明风险评估无法为转基因植物应用提供足够的安全保障,还必须通过开展系统而长期的环境监测,明确转基因植物在生产应用后的实际环境影响。联合国环境规划署和欧盟等已经制定了转基因植物环境监测的法规和技术指南,一些国家实施了系统的转基因植物环境监测。对转基因植物所产生的环境风险以及环境监测应包括的内容进行了综述。     

转基因动物在生物制药工业中的应用

简要论述了转基因动物的概念、制作方法及应用领域,回顾了转基因动物技术的发展及现状,分析了转基因动物与克隆动物的区别.就转基因动物在制药工业和生物医药领域中的国内外研究与开发应用情况进行了阐述,同时展望了转基因动物制药的发展前景及对社会的影响.     

Feeding behaviour of Helicoverpa armigera larvae on insect-resistant transgenic cotton and non-transgenic cotton

Abstract: Feeding behaviour of Helicoverpa armigera Hübner (Lep.; Noctuidae) larvae on non‐transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum L.), Zhong 30, and transgenic cowpea trypsin inhibitor (CpTI)‐Bt cotton, SGK 321, and non‐transgenic cotton, Shiyuan 321, was investigated in both choice tests and no‐choice tests. The results of choice tests suggested that neonates have the ability to detect and avoid transgenic cotton. In the choice tests of neonates with both transgenic and non‐transgenic cotton leaves, a significantly greater proportion of larvae and higher consumption were observed on non‐transgenic cotton than on the transgenic Bt or CpTI‐Bt cotton. In the choice tests with leaves of two transgenic cotton lines, the proportion of neonates on leaf discs of the two lines was not significantly different, but there was significantly higher consumption on CpTI‐Bt transgenic cotton than that on Bt transgenic cotton. In addition, significantly more neonates were found away from the leaf discs, lower consumption and higher mortality were achieved in the choice test with two transgenic cotton leaves than in the choice tests containing non‐transgenic cotton leaves. Leaves and buds were examined in choice tests of fourth instars. It appeared that fourth instars were found in equal numbers on transgenic and non‐transgenic cotton, except when larvae were exposed to leaves for 3 h. However, the total consumption on transgenic cotton was lower than that of the non‐transgenic cotton, so fourth instars may still have the capacity to detect transgenic cotton and reduce feeding on it, although they showed no preference on either transgenic or non‐transgenic cotton. More larvae were found off diet in the treatments with leaves than that of buds, and the number of injured leaf discs by per fourth instar was significantly higher than that of buds in choice tests, suggesting that leaf is a less preferred organ for H. armigera larvae, elicited more larval movements. Similarly, in no‐choice tests of fifth instars, significantly fewer feeding time and more moving time occurred on leaf than that of bud, boll and petal. When cotton line was considered, compared with non‐transgenic cotton, significantly lower feeding time and higher resting time occurred on the two transgenic cottons. Overall, H. armigera larvae have the ability to detect the transgenic Bt and CpTI‐Bt cottons or the less preferred organs and selectively feed more on the non‐transgenic cotton or the preferred organs, especially the neonates, which have a high capacity for avoiding transgenic cotton.     

Effects of Transgenic Petunia Expressing Bacillus thuringiensis Toxin on Selected Lepidopteran Pests

Toxicity of 11 transgenic petunia lines expressing the CryIA (c) insecticidal crystal protein of Bacillus thuringiensis subsp . kurstaki was investigated using lepidopteran neonates Spodoptera exigua, Trichoplusia ni and Manduca sexta. Mortality of S. exigua, T. ni and M. sexta varied within and among transgenic petunia lines . Bioassay results demonstrated that levels of CryIA (c) expression obtained in 7 out of the 11 transgenic petunia lines produced at least 50% mortality in S. exigua and T. ni, and all 11 transgenic lines produced more than 80% mortality in M. sexta. Demographic analysis of the biological impact of transgenic petunia on S. exigua revealed that sub - lethal feeding on transgenic petunia significantly reduced larval weight and prolonged larval and pupal development times . Continuous feeding on transgenic petunia significantly reduced lifetime fecundity , egg hatch and longevity in female and male moths . Compared with insects fed continuously on non - transgenic petunia , lifetime fecundity and net reproductive rate were reduced by 58 and 84% in insects fed continuously on transgenic petunia respectively . Mean generation time was 8 days longer for insects fed continuously on transgenic petunia than for insects fed on non - transgenic petunia . Ovipositional attractiveness of transgenic petunia to S. exigua with respect to non - transgenic tomato or lettuce plants was similar , suggesting that petunia / tomato and petunia / lettuce may not be effective trap - cropping combinations . The potential and implications of using transgenic petunia as trap plants interplanted with crop plants for management of lepidopteran pests in the field are discussed .     

提高转基因表达水平的策略

随着转基因相关技术的发展,转基因动物技术在许多方面得到了成功应用.但外源基因在体内的表达仍然难以预测,特别是大动物的转基因,由于制备效率低下,因而难以筛选出足够的高表达的阳性动物数.基因表达调控研究对提高外源基因在动物体内的表达水平提供了一些新手段,本就避免转基因的位置效应、控制外源基因在动物宿主基因组中的整合、提高转基因的表达效率、构建转基因载体和使用外源基因需要注意的问题等进行综述.     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

转基因水稻的研究和应用

植物基因工程的兴起,使特定的外源基因引入植物细胞成为可能。水稻转基因研究是国内外植物分子遗传学研究的热点之一。近十几年来,水稻转基因研究已取得显著进展。综述了水稻基因转化的方法、转基因技术在水稻上的应用及外源基因在转基因后代中的遗传表达的研究进展。     

转基因水稻对土壤微生物群落结构及功能的影响

以非转基因水稻为对照,以变性梯度凝胶电泳(DGGE)和Biolog技术为手段,研究了2种转基因水稻对土壤微生物群落结构及功能的影响。结果显示:转基因水稻仅在生长发育旺盛期对土壤细菌数量有显著影响;且不同品种转基因水稻土壤微生物间的遗传距离大于转基因水稻与对照间土壤微生物的距离,即2个转基因水稻品种对土壤微生物群落遗传多样性的影响均不显著;在水稻抽穗期,2种转基因水稻与其对照的土壤微生物群落在72h时的平均光密度呈现显著差异,而到了成熟期则差异不显著。土壤微生物群落多样性指数和均匀度指数也表现出类似趋势。本试验证明,在水稻生长发育旺盛时期,Mclntosh指数(u)是一个有效区分转基因水稻和非转基因水稻土壤微生物群落多样性的指标。     

转基因植物外源基因逃逸的途径

随着转基因植物商品化进程的加快,对其进行生态风险性评价也日益引起许多学者和科学家的重视。人们对转基因逃逸到其它亲缘物种中能否产生超级杂草、昆虫是否会产生耐受性及转基因植物对生物多样性的影响等问题进行了广泛的研究。本文对转基因植物中外源基因逃逸的几种主要途径作了综述,并对研究方法与手段进行了简要讨论,希望能为有关决策和科研部门提供一些思路。     

Age-specific bioassays and fecundity of Phthorimaea operculella (Lepidoptera: Gelechiidae) reared on cry1Ac -transgenic potato plants

Variation in the susceptibility of lepidopterous pest larvae of different ages to transgenic crops and the potential for survivors to reproduce could have important consequences for the development of resistance in such pests. Experiments were undertaken in the laboratory to determine if larvae of the potato tuber moth, Phthorimaea operculella, of different ages (0 (< 1 day old), 3, 5, 7 days) varied in their susceptibility to cry1Ac9–transgenic potato (Solanum tuberosum) foliage grown in the glasshouse or field. The survival and fecundity of larvae reared on transgenic tubers was also determined in the laboratory. There were no apparent differences in susceptibility of larvae of different ages to transgenic foliage. Larvae fed glasshouse or field‐grown non‐transgenic foliage had significantly larger relative growth indices and more larvae pupated, than those fed transgenic foliage, regardless of larval age. Eggs from a laboratory colony were placed on transgenic or non‐transgenic tubers to measure survival and fecundity. Between 6% and 15% of eggs placed on transgenic tubers developed into pupae for three of the four transgenic potato lines tested. On one transgenic line, only six adults emerged from 1300 eggs. In contrast, between 71% and 97% of the eggs placed on non‐transgenic tubers developed into pupae. Male and female pupae from transgenic lines weighed less than those from non‐transgenic lines. The fecundity of females from two of four transgenic lines was lower than from the non‐transgenic parent cultivar. Although larvae of different ages did not exhibit any overall age‐dependent pattern of increasing or decreasing susceptibility to transgenic foliage of glasshouse or field‐grown plants, the ability of larvae to survive and reproduce on transgenic tubers suggests this pest has the ability to evolve resistance to the transgenic plants used in the present study.     

转基因植物外源基因逃逸的途径

随着转基因植物商品化进程的加快,对其进行生态风险性评价也日益引起许多学者和科学家的重视,人们对转基因逃逸到其它亲缘物种中能否产生超级杂草,昆虫是否会产生耐受性及转基因植物对生物多样性的影响等问题进行了广泛的研究,本文对转基因植物外中源基因逃逸的几种主要途径作了综述,并对研究方法与手段进行了简要讨论,希望能为有关决策和科研部门提供一些思路。     

Disease resistance, stress response and effects of triploidy in growth hormone transgenic coho salmon

Diploid and triploid coho salmon Oncorhynchus kisutch transgenic for growth hormone (GH) and control coho salmon were compared for differences in disease resistance and stress response. Resistance to the bacterial pathogen Vibrio anguillarum was not affected in transgenic fish relative to their non‐transgenic counterparts when they were infected at the fry stage, but was lower in transgenic fish when infected near smolting. Vaccination against vibriosis provided equal protection to both transgenic and non‐transgenic fish. Triploid fish showed a lower resistance to vibriosis than their diploid counterparts. Diploid transgenic fish and non‐transgenic fish appeared to show similar physiological and cellular stress responses to a heat shock. These studies provide information useful for both performance and ecological risk assessments of growth‐accelerated coho salmon.     

转基因植物检测技术的研究进展

现代植物基因工程使转基因植物及其产品越来越多地进入人们的生活,转基因植物安全性在世界范围内引起了广泛关注,对转基因植物检测技术的需求也越来越紧迫。就转基因植物检测技术的研究进展进行综述,重点介绍以基因和蛋白为目标的检测技术,包括PCR、ELISA和基因芯片技术的最新进展,并对不同方法的优缺点进行对比。此外,提出对特定代谢产物的检测是转基因植物检测的重要组成部分,是以后检测技术的发展趋势之一。最后,以差异蛋白为检测目标,结合研究工作提出基于双向电泳技术的转基因植物检测方法及其产品溯源方案。     

草坪草育种研究进展 (综述)

介绍草坪草传统育种和转基因育种的历史与现状,概述草坪转基因育种的操作过程,对植物特别是草坪草转基因过程中存在的一些问题如基因丢失,基因沉默,基因流引起的转基因安全性等进行评述。     

Efficiency of human lactoferrin transgenic donor cell preparation for SCNT

The combination of somatic cell nuclear transfer (SCNT) and transgenic technology leads to the production of transgenic cloned animals, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. The purpose of this study was to establish an efficient procedure to prepare human lactoferrin (hLTF) transgenic donor cells for SCNT. Thus, two cell culture systems were employed: caprine mammary epithelial cells (for evaluation of the hTLF transgenic expression in vitro), and fetal-derived fibroblast cells (for identification of competent transgenic donor cells). Induced by hormonal signals, recombinant hLTF was detected in the supernatant of transfected mammary epithelial cells by Western blot. Reliable hLTF transgenic fibroblast cell clones were identified by screening with multiple PCR amplification, EGFP fluorescence, and chromosomal counting (32.5+/-2.3%). This study may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant pharmaceuticals from the milk of transgenic animals.     

心脏特异表达人源FAM55A转基因小鼠的建立

目的建立心脏特异表达的人源FAM55A转基因小鼠,为研究该基因在心肌病发病中的作用提供模型。方法 Western blot检测FAM55A在野生型小鼠与cTnTR141W转基因小鼠心脏组织中的表达变化及其在野生小鼠的组织表达谱。克隆人源FAM55A基因入α-MHC启动子下游构建a-MHC-FAM55A表达载体,显微注射法建立FAM55A转基因小鼠。PCR鉴定转基因首建鼠的基因型。Western blot鉴定人源FAM55A在转基因小鼠心脏中的表达,超声检测转基因小鼠心脏的几何构型和功能。HE染色检测转基因小鼠心脏的病理改变。结果 FAM55A在野生型小鼠心脏中有少量表达,在扩张型心肌病小鼠的心脏中表达增加。建立了1个心脏组织特异表达人源FAM55A转基因小鼠品系。与野生型小鼠相比,FAM55A转基因小鼠的心脏收缩期和舒张期左室前壁从1月龄到5月龄持续增厚,3月龄转基因小鼠心脏射血分数和短轴缩短率稍有增强,1月龄和5月龄转基因小鼠心脏功能则与同龄野生型小鼠相比无变化。组织学检测显示,转基因小鼠心脏左室心肌细胞不均匀肥大,但不发生紊乱。结论 FAM55A在扩张型心肌病小鼠的心脏中表达上调,建立了心脏特异表达的人源FAM55A转基因小鼠,为进一步和心肌病小鼠模型杂交,研究该基因在心肌病发病中的作用提供了工具。