家畜转基因育种研究进展

转基因技术可以将外源基因导入家畜基因组,使其获得新的可遗传性状,为培育优良家畜品种提供了革命性途径。DNA显微注射法和体细胞克隆法是制备转基因家畜最常用的方法。应用转基因技术可以进行家畜抗病育种(抗病毒、抗菌和抗寄生虫),改良家畜的生产性状(胴体组成、奶品质、产毛、繁殖力和生长速度),培育环保型家畜新品种。文章从动物转基因技术入手,阐明其在家畜品种改良方面的研究现状和策略,并探讨家畜转基因育种面临的问题和应用前景。     

转基因家畜对环境和食品的安全性分析及应对策略

应用转基因技术对家畜进行遗传改良,必将给畜牧业生产带来革命性的变化。与此同时,与其它转基因生物一样,其安全性也引起了广泛关注。为促进转基因家畜研发工作的健康发展,对其可能影响环境和食品安全性的因素进行了分析,并提出相应的应对策略和技术措施,供参考。     

人乳铁蛋白在转基因马铃薯块茎中的表达

人乳铁蛋白(human lactoferrin,hLF)是人体非特异性免疫系统的重要成员之一,具有抗细菌、真菌和抗病毒活性及其他多种功能.报道将hLF基因的cDNA与马铃薯(Solarium tuberosum L.)块茎专一性表达patain基因启动子融合后通过农杆菌介导导入马铃薯,PCR检测证实获得了多个转基因株系,RT-PCR阳性结果说明hLF mRNA在马铃薯植株中得到了表达.同时,经过ELISA及Western blot检测证实,转基因马铃薯表达了hLF并具有人乳铁蛋白的活性.     

植物铁蛋白转基因的应用

植物铁蛋白是一种铁存放蛋白,既可以储存大量的可被植物利用的铁,又能抵抗环境胁迫。利用铁蛋白的转基因研究不仅可以缓解由于铁缺乏而引起的一系列疾病,而且能提高植物对环境的耐受性,在生物治疗中具有重要意义。文章就铁蛋白的结构以及铁蛋白转基因植物在上述几个领域中的研究进展作简单的介绍。     

人乳铁蛋白 (hLF) 转基因山羊的遗传背景分析

以随机整合方式获得的转基因动物外源基因的拷贝数、整合位点及染色体核型等遗传背景并不清楚,可能会存在外源基因的沉默整合、无效整合、毒性整合以及其表达水平不可预测等问题。文中选取了6只原代(F0)及其相对应的子一代(F1)的人乳铁蛋白(hLF)转基因山羊作为研究对象,分别颈静脉采血、提取DNA,通过染色体核型分析、实时荧光定量PCR(qPCR)、ELISA和Westernblotting等检测技术,研究其外源基因的遗传背景与表达水平。结果显示,6只F0代转基因山羊的染色体没有明显的形态变异、数量改变等异常情况。相对拷贝数高低不同(2–16),且能够稳定地遗传给下一代,F0和F1代hLF基因拷贝数一致。F1代转基因山羊表达hLF水平最高可达1.12 g/L(L3-1,拷贝数8)。结果表明,整合的外源基因能够稳定地遗传下一代,也没有对转基因山羊个体的生长发育造成障碍,而且拷贝数高低与hLF表达水平无明显的相关性,这为转基因山羊及其他转基因动物的新品种培育奠定了基础,解析了遗传背景。     

人乳铁蛋白基因在烟草中的表面及抗细菌与病毒的能力

从人血液中性白细胞中分离细胞总ＲＮＡ,用ｏｌｉｇｏ（ｄＴ）为引物进行ｃＤＮＡ第１条链合成,再用乳铁蛋白ｃＤＮＡ的特异性此物进行聚合酶链反应（ＰＣＲ）,分两２段合成并拼成完整的乳铁蛋白ｃＤＮＡ,核苷酸序列测定证明,该ｃＤＮＡ所编码的氨基酸与前人论证的人乳铁蛋白的氨基酸序列完全一致,将该ｃＤＮＡ构建成植物表达载体ｐ３５ＳｈＬＦｃ,经根癌农杆菌ＬＢＡ４４０４感染烟草叶盘,获得具有卡那霉素抗性的烟草植株,     

牛乳铁蛋白肽转基因小鼠的构建

目的:探讨牛乳铁蛋白肽在转基因鼠乳汁中的表达及其抑菌活性。方法:将实验室构建并保存的包含山羊β-酪蛋白基因启动子和牛乳铁蛋白肽基因的乳腺特异表达载体PI-bcp-LfcinB,用Xho I和Nru I双酶切,得到含有全部表达盒的显微注射DNA片段,采用常规显微注射技术获得转基因小鼠。并通过对泌乳期转基因雌鼠乳腺组织的RT-PCR检测,确定了牛乳铁蛋白肽在mRNA水平的表达,同时利用琼脂板扩散法检测了转基因鼠乳汁中表达产物的抑菌活性。结果:获得了牛乳铁蛋白肽转基因小鼠,且转基因小鼠乳汁中能够表达具有抑菌活性的牛乳铁蛋白肽。结论:通过转基因动物乳腺可以获得具有生物活性的牛乳铁蛋白肽,为进一步研究抗菌肽转基因牛、培育抗乳房炎奶牛新品种以及通过建立转基因动物生物反应器进行抗菌肽的大量生产奠定了基础。     

用转基因技术改良家畜还很困难

<正> 澳大利亚畜产部首席技术官、澳大利亚联帮科学与工业研究组织(CSIRO)的 JamesD.Murray 说:\"要商品化转基因家畜看来至少还要10年时间\"。Murray 研究小组研究了将基因导入绵羊,以提高生长激素的浓度问题。但是得出的结果和预期的有相当大的距     

含转人乳铁蛋白奶粉对SD大鼠亚慢性毒性的研究

目的:研究人乳铁蛋白奶粉对SD大鼠营养状况、生理、生化指标等亚慢性毒性的影响.方法:取初断乳SD大鼠140只,雌雄各半按照体重随机分为7组.分别饲喂添加比例为7.5%,15%和30%转基因成分奶粉的饲料、添加比例为7.5%,15%和30%不舍转基因成分奶粉的饲料和基础饲料90天.在实验中期和未期采血检测血液常规指标.实验结束时处死实验动物,计算脏器指数,并对主要脏器进行病理组织学观察.结果:试验期间各组动物生长发育情况良好,给予含转基因成分奶粉实验组动物的血常规与不含转基因成分奶粉组和常规饲料组相比均无显著性差异(P≤0.05),未见各组实验动物的脏器组织出现任何病理形态学改变.结论:未发现含转基因成分的奶粉对实验动物有亚慢性毒性.     

外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

Irregular patterns of transgene silencing in allohexaploid oat

An irregular pattern of transgene silencing was revealed in expression and inheritance studies conducted over multiple generations following transgene introduction by microprojectile bombardment of allohexaploid cultivated oat (Avena sativa L.). Expression of two transgenes, bar and uidA, delivered on the same plasmid was investigated in 23 transgenic oat lines. Twenty-one transgenic lines, each derived from an independently selected transformed tissue culture, showed expression of both bar and uidA while two lines expressed only bar. The relationship of the transgenic phenotypes to the presence of the transgenes in the study was determined using (1) phenotypic scoring combined with Southern blot analyses of progeny, (2) coexpression of the two transgenic phenotypes since the two transgenes always cosegregated, and (3) reactivation of a transgenic phenotype in self-pollinated progenies of transgenic plants that did not exhibit a transgenic phenotype. Transgene silencing was observed in 19 of the 23 transgenic lines and resulted in distorted segregation of transgenic phenotypes in 10 lines. Silencing and inheritance distortions were irregular and unpredictable. They were often reversible in a subsequent generation of self-pollinated progeny and abnormally segregating progenies were as likely to trace back to parents that exhibited normal segregation in a previous generation as to parents showing segregation distortions. Possible causes of the irregular patterns of transgene silencing are discussed.     

转基因植物的研究概况及应用领域

就转基因技术在作物育种上的研究、应用状况、应用领域以及产生的经济、社会和环境效益几方面进行了概述。     

Microbiological quality of raw milk produced in Estonia

Aims: The microbial quality of farm bulk‐tank raw milk produced in Estonia during years 2004–2007 was investigated. Methods and Results: Bulk‐tank milk samples were analysed for lactic acid bacteria count (LABC), psychrotrophic bacteria count (PBC), aerobic spore‐forming bacteria count (ASFBC), total bacterial counts using BactoScan and somatic cell count (SCC) using Fossomatic. Randomly selected psychrotrophic isolates were subjected to 16S–23S PCR‐ribotyping. LABC remained below 10⁴ CFU ml^?1 in most samples, while psychrotrophic micro‐organisms dominated in 60% of farms. PBC ranged from 4·2 × 10² to 6·4 × 10⁴ CFU ml^?1, and ASFBC varied from 5 to 836 CFU ml^?1. Conclusions: In general, the microbiological quality of the farm bulk‐tank milk was good – more than 91% of samples contained <50 000 CFU ml^?1, and SCC in the majority of samples did not exceed the internationally recommended limits. Genus Pseudomonas spp. was the dominating spoilage flora with Pseudomonas fluorescens as the prevailing species. Significance and Impact of the Study: Specific bacterial groups (LABC, PBC and ASFBC), not analysed routinely by dairies, were determined in bulk‐tank raw milk of numerous dairy farms during 4‐year period. Based on the survey, dairy plants can better control their supply chains and select farms (milk) for the production of specific products, i.e. milk with low PBC and high LABC for cheesemaking.     

Rapid PCR-based determination of transgene copy number in rice

We present a simple, rapid, and low-cost method to determine transgene copy number in rice. More than 100 first- and second-generation transgenic rice plants were tested. The plasmid (pRCopy) used for rice transformation contains the specific gene of interest and a partially deleted cytochrome c gene (cyc), a single-copy gene in rice. A 132-bp segment of the cloned ricecyc was shortened to 108 bp by deleting a 24-bp internal fragment. After PCR amplification of the genomic DNA from transgenic rice harboring pRCopy, the 2 expected bands were found. The 121-bp band corresponds to the endogenouscyc; the 97-bp band comes from the integrated pRCopy. Clear distinctions can be made between single and multiple copies of the transgene by comparing band densities.     

Selenium content of milk and milk products of Turkey. II

Selenium content of 1028 milk and milk products of Turkey are presented in this study. The selenium content of human milk (colostrum, transitional, and mature milk), various kinds of milk [cow, sheep, goat, buffalo, paper boxes (3%, 1.5%, 0.012% fat), bottled milk, condensed milk (10% fat), mineral added milk (1.6%), and banana, strawberry, and chocolate milk] and milk products (kefir, yogurt, Ayran, various cheese, coffee cream, ice cream, butter, margarine, milk powder, and fruit yogurt) in Turkey were determined by a spectrofluorometric method. The selenium levels of cow milks collected from 57 cities in Turkey were also determined. Selenium levels in cow milk varied with geographical location in Turkey and were found to be lowest for Van and highest for Aksaray. The results [milk (cow, sheep, goat, buffalo and human) and milks products] were compared with literature data from different countries.     

土壤植物营养与农产品品质及人畜健康关系

综述了土壤中Ca,Mg,S,Zn,Fe等中微量营养元素及Se,I等有益元素的丰缺情况,概述了矿质营养元素和有机物质对农产品品质的影响与人畜健康的关系,展望了今后应预以加强的研究方向,为进一步开展土壤植物营养与产品品质的研究提供参考。     

Chromosomal localization of a proinsulin transgene in Japanese quail by laser pressure catapulting

Transgenic avian bioreactors produce therapeutic recombinant proteins in egg white. To date, however, methods for transgenic modification of the avian genome or determining transgenic status of individual birds are scarce. The dual, but interrelated, goals of this research were to: (1) develop a method of detecting stable DNA insertion into Japanese quail; and (2) provide a method for gene location on avian chromosomes. We created Teflon-coated coverslip slides to facilitate laser pressure catapulting of avian chromosomes for DNA amplification and nucleotide sequencing. Transgenic G₂ Japanese quail, containing germline incorporation of proinsulin, were identified by isolation of chromosomes using laser microdissection and laser pressure catapulting. Subsequent amplification of each chromosome identified 2–5 chromosomes with the proinsulin transgene inserted. Nucleotide sequencing of each chromosomal insertion was identical to the proinsulin portion of the original vector. By applying laser pressure catapulting and PCR of individual chromosomes, we were able to determine that the transgene correctly inserted into avian chromosomes and that the majority of the insertions occurred within microchromosomes. Because many potential therapeutic transgenes have similar or nearly identical nucleotide sequence to the host’s native gene, laser microdissection and subsequent analysis may be required for detailed documentation of transgene expression before proceeding with transgenic protein production.