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。     

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.     

家畜的遗传工程

人们希望家畜的遗传工程能在农业生产方面产生较大的影响。但如果不经过多代的研究,不可能准确地估价转基因表达的结果。本文以两种转基因猪系为对象,系统地研究了因转基因而使其血液中生长激素(bGH)水平长期升高所产生的有利和不利的结果。连续两代表达bGH基因的猪在每日增重及食物利用率方面都有明显改进,而且身体组成也有变化,如皮下脂肪有明显减少。     

Evaluation of quality changes in udder quarter milk from cows with low-to-moderate somatic cell counts

Much emphasis has been put on evaluating alterations in milk composition caused by clinical and subclinical mastitis. However, little is known about changes in milk composition during subclinical mastitis in individual udder quarters with a low-to-moderate increase in milk somatic cell count (SCC). This information is needed to decide whether milk from individual udder quarters with a moderate-to-high increase in milk SCC should be separated or not. The aim of this study was to determine how milk composition in separate udder quarters is affected when cow composite milk has low or moderately increased SCC levels. Udder quarter and cow composite milk samples were collected from 17 cows on one occasion. Milk yield was registered and samples were analyzed for SCC, fat, total protein, whey proteins, lactose, citric acid, non-protein nitrogen (NPN), lactoferrin, protein profile, free fatty acids (FFAs), lactate dehydrogenase (LDH), proteolysis, sodium and potassium. Bacteriological samples were collected twice from all four quarters of all cows. The cows were divided into three groups depending on their SCC at udder quarter level. The first group comprised healthy cows with four udder quarters with low SCC, <50 000 cells/ml; composition was equal when opposite rear and front quarters were compared. In the second and the third groups, cows had one udder quarter with 101 000 cells/ml < SCC < 600 000 cells/ml and SCC > 700 000 cells/ml, respectively. The remaining udder quarters of these cows had low SCC (<100 000 cells/ml). Despite the relatively low average cow composite SCC = 100 000 cells/ml of Group 2, milk from affected udder quarters exhibited lower casein number, content of lactose and β-casein (β-CN), while the content of whey protein, sodium, LDH and α-lactoalbumin (α-la) were higher compared to healthy opposite quarters. In addition to these changes, milk from affected udder quarters in Group 3 also exhibited lower values of potassium and αs1-casein (αs1-CN) and higher values of lactoferrin when compared to milk from opposite healthy quarters. This indicates that even when the SCC in cow composite milk is low, there might exist individual quarters for which milk composition is changed and milk quality impaired.     

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。     

Feeding heat-oxidized oil to dairy cows affects milk fat nutritional quality

Heating oil and oilseeds results in oxidation products that affect ruminal biohydrogenation of polyunsaturated fatty acids, altering milk fatty acids profile, and could be transferred to milk. An experiment was conducted to investigate the effects of oil heating on rumen and milk fatty acids profile and the transfer of oxidation products to milk. Sunflower oil was heated at 150°C for 15 h and given to lactating dairy cows in a 2×2 arrangement: two groups of two cows, equipped with a ruminal cannula and receiving two diets (containing either heated or unheated oil) during two experimental periods. Oil heating generated hydroperoxides and/or hydroxyacids and aldehydes, in particular trans-2,trans-4-decadienal. In milk, heated oil only significantly decreased trans-11-C18:1 and cis-9,trans-11-CLA percentage compared to non-heated oil, and slightly increased cis-9,cis-12-C18:2 percentage, which was probably linked to an inhibition of the ruminal Δ12 isomerase by oxidative products in the rumen. However, feeding highly oxidized oil did not result in the appearance of hydroperoxides or hydroxyacids in milk and did not increase milk aldehydes content.     

畜禽粪便施用对稻麦轮作土壤质量的影响

通过采集试验区长期施用鸡粪 (PL)、猪粪 (LM) 和化肥 (CF) 的稻麦轮作耕层和犁底层土壤,分析了不同施肥处理土壤有机碳和养分含量、土壤物理结构特征、土壤生物学性质的差异,探讨了长期施用畜禽粪便对土壤质量的影响。研究结果显示,长期施用畜禽粪便耕层和犁底层土壤有机碳含量显著高于施用化肥处理(P<0.05);与CF处理比较,PL和LM处理土壤氮、磷、钾全量和有效养分含量均明显增加,其中耕层土壤Olsen-P 含量为施用化肥处理的7-8倍,速效钾含量比施用化肥土壤高89.2%-102.9%。施用畜禽粪便明显改善了土壤物理结构,其耕层土壤大孔隙体积、中孔隙体积和总孔隙度分别为CF处理土壤的1.48-1.70倍,1.35-1.75倍和1.07-1.11倍;土壤团聚体水稳定性显著增强,而土壤抗张强度显著降低。施用畜禽粪便土壤微生物和生化性质也明显高于施用化肥土壤,其中LM处理耕层土壤MBC和MBN最大,分别是CF处理土壤的2.1倍和1.5倍;施用畜禽粪便土壤脲酶和转化酶活性也分别为施用化肥土壤的3.5-6.7倍和1.6-2.1倍。相关分析显示,土壤有机碳含量与各肥力指标间均表现出极显著相关(P<0.01)。研究结果说明,长期施用畜禽粪便土壤质量显著高于仅施化肥土壤。     

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.     

Re-defining efficiency of feed use by livestock

Livestock, particularly ruminants, can eat a wider range of biomass than humans. In the drive for greater efficiency, intensive systems of livestock production have evolved to compete with humans for high-energy crops such as cereals. Feeds consumed by livestock were analysed in terms of the quantities used and efficiency of conversion of grassland, human-edible ('edible') crops and crop by-products into milk, meat and eggs, using the United Kingdom as an example of a developed livestock industry. Some 42 million tonnes of forage dry matter were consumed from 2008 to 2009 by the UK ruminant livestock population of which 0.7 was grazed pasture and 0.3 million tonnes was conserved forage. In addition, almost 13 million tonnes of raw material concentrate feeds were used in the UK animal feed industry from 2008 to 2009 of which cereal grains comprised 5.3 and soyabean meal 1.9 million tonnes. The proportion of edible feed in typical UK concentrate formulations ranged from 0.36 for milk production to 0.75 for poultry meat production. Example systems of livestock production were used to calculate feed conversion ratios (FCR - feed input per unit of fresh product). FCR for concentrate feeds was lowest for milk at 0.27 and for the meat systems ranged from 2.3 for poultry meat to 8.8 for cereal beef. Differences in FCR between systems of meat production were smaller when efficiency was calculated on an edible input/output basis, where spring-calving/grass finishing upland suckler beef and lowland lamb production were more efficient than pig and poultry meat production. With the exception of milk and upland suckler beef, FCR for edible feed protein into edible animal protein were >1.0. Edible protein/animal protein FCR of 1.0 may be possible by replacing cereal grain and soyabean meal with cereal by-products in concentrate formulations. It is concluded that by accounting for the proportions of human-edible and inedible feeds used in typical livestock production systems, a more realistic estimate of efficiency can be made for comparisons between systems.     

Udder quarter milk composition at different levels of somatic cell count in cow composite milk

Automatic milking systems have made possible the separation of high- and low-quality milk at the udder quarter level during the milking process. The aim of this study was to investigate the composition and yield of milk from individual udder quarters to determine whether deteriorated milk composition occurs in udders that are assumed to be healthy and whether quarters with high-quality milk are found in udders with high milk somatic cell count (SCC). Milk samples were collected on one occasion from 90 cows at udder quarter level and cow composite level. The milk was analyzed for content of total protein, whey protein, casein, fat, lactose, citric acid and SCC; milk yield was registered. The cows were divided into three groups depending on the SCC of their composite milk. Cows in group 1, cow composite SCC < 100 000 cells/ml, were assumed to have healthy udders. However, instances of increased SCC and decreased milk quality were discovered in one or more udder quarters of approximately 30% of the group. Cows in group 2, cow composite SCC of 100 000 to 300 000 cells/ml, and group 3, cow composite SCC > 300 000 cells/ml, were assumed to have affected udders. However, the majority of these cows had one or more udder quarters in which increased SCC and deteriorated milk quality were not detected. Calculations of bulk-tank milk values, when separation of milk from affected udder quarters was performed, indicate that SCC changes to a much greater degree compared to the other milk components. These results show that milk from affected udder quarters suffers compositional changes, but calculations of simulated separation indicate that the compositional changes in bulk-tank milk are small. The effect of separation of milk from individual udder quarters on bulk-tank milk needs to be further studied.