转基因烟草荧光定量检测方法研究

依据实时定量PCR原理,参照35S启动子、NOS终止子、GUS基因和NPTII基因序列设计TaqMan引物和荧光标记探针。采用美国MJ公司OpticonTM2荧光定量PCR检测系统对烤后烟叶进行转基因定量检测技术研究,从中筛选出扩增效率高,灵敏度好的PCR引物和探针序列,同时通过对扩增体系,扩增条件的梯度实验,优化出荧光定量检测的最佳反应体系和反应条件,从而建立了转基因烟草定量检测方法。该方法经验证其检测灵敏度达到0.05％。在2003年6月参加CORESTA(国际烟草科研与合作中心)组织的国际烟草转基因定量检测合作试验中,该优化转基因烟草定量检测技术获得了较好成绩,对盲检样品检测结果评价(Z-score)列国际12家实验室之首,证明此法灵敏度高、稳定性好。     

烟草环斑病毒的定量检测技术研究

目的:建立特异、灵敏、快速的TaqMam实时荧光定量PCR方法,用于烟草环斑病毒(TRSV)的定量检测。方法:用纳米磁珠法提取病毒RNA,构建包含烟草环斑病毒全CP序列的质粒标准品。根据CP保守序列设计特异性的引物和TaqMam荧光探针,构建标准曲线,建立TRSV的实时荧光绝对定量PCR方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:建立的方法特异性好,与南芥菜花叶病毒、马铃薯X病毒和马铃薯Y病毒均无交叉反应;至少能检测到767个病毒拷贝,灵敏度比普通PCR高100倍;同一样品试验内及试验间重复性实验的变异系数均小于3%,重复性好;检测结果准确可靠,构建的标准曲线有较好的线性关系(R2=0.997)。结论:建立的TRSV TaqMan实时荧光定量PCR检测方法可满足口岸高通量、快速、准确的检验检疫要求。     

转基因植物核酸成分检测技术研究进展

首先对转基因核酸成分检测的靶序列特征进行了阐述,对转基因植物核酸成分的定性、定量检测技术研究进展进行了综述,包括基于PCR的检测技术、基于等温核酸扩增的检测技术、基因芯片检测技术、基于高通量测序和新型转基因核酸检测技术(如生物传感器技术、毛细管电泳技术和纳米刻度技术等),重点介绍了各种检测技术的原理、特点、研究现状和发展动态,并对各种方法的优缺点进行了比较。     

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。     

转基因产品检测方法概述

随着转基因技术的快速发展,转基因生物及其产品日益增多,但其安全性问题引起了国际社会的广泛关注。转基因产品的检测已纳入国内外检验检疫部门的检测项目,采用的检测方法是建立在已商品化生产的转基因生物外源基因的构建及表达情况的基础上的,包括蛋白质检测和DNA检测方法。蛋白质检测方法有ELISA、试纸条、免疫PCR等,DNA检测方法有PCR、多重PCR、PCR-EUSA、PCR-GeneScan、荧光定量PCR、基因芯片等。     

转基因植物核酸检测策略与体外扩增技术研究进展

转基因生物(Genetically modified organisms,GMOs)特别是转基因作物的商品化应用在世界范围内迅猛发展,在推动农业、医药和工业等领域的飞速发展的同时也逐步吸引越来越多公众注意力,生物安全问题成为突出的争论话题.转基因作物及其产品的快速准确检测成为社会发展的急切需求,转基因技术的多样化也要求检测策略和检测技术的不断改进.系统综述针对各种作物转基因技术和转基因产品的检测策略和技术,全面比较各种技术的特点和适用范围,并对目前面临的问题和发展趋势进行分析.     

高度耐盐双价转基因烟草的研究

随着全球性人口的增长和土地退化的加剧,开发利用广阔盐碱地和干旱土地的需要日益迫切。植物生物技术的日臻完善,为培育高效耐盐植物迎来了一丝曙光。在高渗条件下,耐盐的微生物或植物细胞通过增加胞内一些相溶性溶质的浓度来维持渗透压的平衡。这些可溶性溶质包括无机离子、糖类、多元醇、氨基酸和生物碱等。通过基因工程手段,使细胞内积累脯氮酸⑴、甜菜碱⑵、甘露醇⑶、海藻糖⑷,能够不同程度地提高转基因烟草的耐盐性。多元醇含有多个羟基,亲水性能强,能有效维持细胞内水活度。山梨醇、甘露醇等己糖分子结构、理化性质和生理功能相近。故此．我们认为：不同糖醇在转基因烟草中的积累．可能具有协同(或累加)效应,有希望更大地提高植物耐盐性。我们在获得大肠杆菌mtlD基因(编码l-磷酸甘露醇脱氢酶)和gutD基因(编码6-磷酸山梨醇脱氢酶)克隆⑸的基础上,获得了分别表达mtlD和gutD基因的单价转基因烟草,并首次证实了gucD基因的表达,能显著地提高转基因烟草的耐盐性⑹。本文工作进一步报道同时表达大肠杆菌mtlD和gutD基因双价转基因烟草的高效高度耐盐性。     

转基因植物的PCR检测

１９９６—２０００年全球转基因作物的种植面积，由原来的１７０万公顷增到４４２０万公顷，５年间猛增了２５倍。近年来，随着转基因作物的种类和产量的不断增加，其产品的安全性问题，即它对人类健康、环境保护和生态平衡等存在的潜在危害，也越来越多的受到人们的广泛关注。至今，已有２０多个国家已开展了基因工程技术的安全性研究，同时还陆续制定了相关的实验研究、工业化生产和向环境释放等一系列安全准则、条例、法规或法律。１９９８年以来，欧盟规定对含有转基因成分的食品及其它商品必须加注标签。最近，日本、韩国、澳大利亚和…     

实时荧光定量PCR技术及其在植物转基因产品检测中的应用

实时荧光定量PCR技术因其实时、快速、高效和准确定量的优点,已经广泛用于转基因产品定量检测。本文介绍荧光定量探针技术的原理、特点及其在植物转基因产品检测中的应用情况。     

转基因烟草表达霍乱毒素B亚单位的研究

将霍乱毒素Ｂ亚单位（ＣＴＢ）基因克隆到质粒ｐＢｉｎ４３８中,分别构建植物表达载体ｐＢＩ－ＣＴＢ、ｐＢＩ－ＳＰＣＴＢ和ｐＢＩ－ＣＴＢＥＲ。采用叶盘法分别转化烟草Ｋ３２６,各表达载体得到了一批较基因植株。转基因烟草的ＰＣＲ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分析表明ＣＴＢ基因整合到了烟草基因组中。转基因植株的ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ ｂｌｏｔ分析表明ｐＢＩ－ＳＰＣＴＢ和ｐＢＩ－ＣＴＢＥＲ的转基因植株能有效表     

Heat-inducible hygromycin resistance in transgenic tobacco

We have constructed a chimaeric gene consisting of the promoter of the soybean heat shock (hs) gene Gmhsp17,6-L, the coding region of a hygromycin phosphotransferase (hpt) gene, and the termination sequence of the nopaline synthase (nos) gene. This gene fusion was introduced into tobacco by Agrobacterium-mediated gene transfer. Heat-inducible synthesis of mRNA was shown by northern hybridization, and translation of this RNA into a functional protein was indicated by plant growth on hygromycin-containing media in a temperature-dependent fashion. One hour incubation at 40 °C per day, applied for several weeks, was sufficient to express the resistant phenotype in transgenic plants containing the chimaeric hs-hpt gene. These data suggest that the hygromycin resistance gene is functional and faithfully controlled by the soybean hs promoter. The suitability of these transgenic plants for selection of mutations that alter the hs response is discussed.     

Analysis of microspore-specific promoters in transgenic tobacco

In order to modify the early stages of pollen development in a transgenic context microspore-specific promoters are required. We tested two putatively microspore-specific promoters, the Bp4 promoter from rapeseed and the NTM19 promoter from tobacco. Expression of the gus and barnase reporter genes under the control of these two promoters was studied in transgenic tobacco. Contrary to expectations, the Bp4 promoter became active only after the first pollen mitosis, and not in the microspores. The NTM19 promoter turned out to be highly microspore-specific and directed very high levels of gus expression to the unicellular microspores. The NTM19-barnase transgene caused cell-autonomous death at the mid-unicellular microspore stage, whereas Bp4-barnase induced cell ablation of early to mid-bicellular pollen. Both promoter-barnase transgenes did not affect the sporophyte and were inherited through the female germline. These results show that both the NTM19 and Bp4 promoters are expressed only in the male germline, and that the NTM19 promoter is an excellent tool to direct high levels of transgene expression exclusively to the microspores. This may have important biotechnological applications.     

转抗菌肽D烟草对土壤微生物群落的影响

采用RAPD分子标记技术研究了种植转抗菌肽D烟草的土壤环境中微生物群落遗传多样性的变化,同时用传统平板培养法研究了土壤中可培养微生物在数量上的变化。RAPD分析结果表明,转抗菌肽D烟草与非转基因烟草根围微生物的遗传多样性相关指数并没有显著差异。培养计数结果表明,转抗菌肽D烟草与非转基因烟草根围可培养细菌在数量上有极显著差异,可培养真菌数量有显著减少,可培养放线菌的数量没有显著差异。说明转抗菌肽D烟草可能抑制了病原细菌及其根围相关的微生物,但是不影响微生物的遗传多样性。     

Manipulation of monoubiquitin improves salt tolerance in transgenic tobacco

Ubiquitin (Ub) is regarded as a stress protein involved in many stress responses. In this paper, sense and antisense transgenic tobacco plants, as well as the wild type and vector control, were used to study the role of Ub in salt tolerance of plants. In sense Ta-Ub2 transgenic tobacco plants, there was higher expression of Ub protein conjugates than in the wild type and vector control, but the reverse trend was observed in antisense Nt-Ub1 transgenic plants. The germination rate of tobacco seed, growth status and photosynthesis of the tobacco plants suggested that over-expressing Ub promoted the growth of transgenic tobacco plants and enhanced their salt tolerance, but the opposite effect was seen in plants with repressed Ub expression. Changes in antioxidant capacity may be one of the mechanisms underlying Ub-regulated salt tolerance. Furthermore, improved tolerance to a combination of stresses was also observed in the sense transgenic tobacco plants. These findings imply that Ub is involved in the tolerance of plants to abiotic stress.     

转基因抗虫烟草研究进展

烟草为模式植物,也是外源杀虫基因最早转化成功的植物。文章从转Bt内毒素基因,植物凝集素GNA,Plec,AHA基因,蛋白酶抑制剂PIⅠ,PIⅡ,MTI,SKTI基因,昆虫特异性神经毒素基因,几丁质酶基因,畸形细胞分泌蛋白基因以及双抗虫基因等方面综述了转基因抗虫烟草的抗虫性、转基因抗虫烟草的经济性状等,展望了转基因抗虫烟草的研究和应用前景,以期对烟草害虫的治理尤其是对其他转基因抗虫作物的培育和研究有借鉴作用。     

Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants

Monoclonal antibody production from transgenic tobacco plants offers many advantages over other heterologous production systems, creating the prospect of production at a scale that will allow new prophylactic and therapeutic applications in global human and animal health. However, information on the major processing factors to consider for large-scale purification of antibodies from transgenic plants is currently limited, and is in urgent need of attention. The purpose of this project was to investigate methods for the initial extraction of recombinant immunoglobulin G (IgG) antibodies from transgenic tobacco leaf tissue. Three different transgenic plant lines were studied in order to establish the parameters for optimal extraction of monoclonal antibodies that accumulate in the apoplasm, at the plasma membrane or within the endoplasmic reticulum. For each transgenic line, seven techniques for physical extraction were compared. The factors that determine the optimal extraction of antibodies from plants have a direct influence on the initial choice of expression strategy, and so must be considered at an early stage. The use of small-scale techniques that are applicable to large-scale purification was a particularly important consideration. The optimal extraction technique varied with the target location of IgG in the plant cell, and the dependence of antibody yield on the physical extraction methodology employed, the pH of the extraction buffer and the extraction temperature was demonstrated in each case. The addition of detergent to the extraction buffer may improve the yield, but this was found to be dependent on the site of accumulation of IgG within the plant cell.     

Expression of a single-chain Fv antibody fragment specific for the Hepatitis B surface antigen in transgenic tobacco plants

An anti-Hepatitis B virus surface antigen (HBsAg) single chain Fv (scFv) antibody fragment was expressed in Nicotiana tabacum transgenic plants. The 6-histidine tagged scFv was targeted to either the cytosol, apoplast, and vacuole, or for retention in the endoplasmic reticulum. Expression of active scFv was detected by ELISA in fresh leaf material from F1 transgenic plant lines representative of the genetic constructs targeting the antibody fragment to the apoplastic fluid (AF-12, 0.031% of the total soluble protein), vacuole (V-20, 0.032% of the total soluble protein), and endoplasmic reticulum (ER-52, 0.22% of the total soluble protein). No scFv was detected by ELISA or western blot in the plants transformed with the cytosol construct. The biologically active scFv was easily purified (to 94–95% purity) from ER-52 and AF-12 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the ER-52 plant line indicates that 15–20g of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale.     

Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants

Transgenic tobacco plants containing a mouse metallothionein-I (MT-I) gene fused to the cauliflower mosaic virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming tobacco leaf discs with an Agrobacterium tumefaciens strain carrying the chimaeric gene. Transformants were directly selected and rooted on medium containing cadmium and kanamycin. A total of 49 individual transgenic tobacco plants were regenerated. Among them 20% showed a very high expression level and their growth was unaffected by up to 200 M cadmium, whereas the growth of control plants was severely affected leaf chlorosis occurred on medium containing only 10 M cadmium. The concentration of MT-I in leaves of control and transgenic tobacco was determined with Cd/haemoglobin saturation assay, a polarographic method and western blotting. In addition, seeds from self-fertilized transgenic plants were germinated on medium containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The ratio of tolerant to susceptible plants was 3:1 indicating that the metallothionein gene is inherited as a single locus.     

Breakage of transgenic tobacco roots for monoclonal antibody release in an ultra‐scale down shearing device

Transgenic tobacco roots offer a potential alternative to leaves for monoclonal antibody (MAb) production. A possible method for extraction of MAbs from roots is by homogenization, breaking the roots into fragments to release the antibody. This process was assessed by shearing 10 mm root sections (“roots”) in a 24 mL ultra‐scale down shearing device, including an impeller with serrated blade edges, intended to mimic the action of a large‐scale homogenizer. Size distributions of the remaining intact roots and root fragments were obtained as a function of shearing time. The data suggest that about 36% of the roots could not be broken under the prevailing conditions and, beyond these unbreakable roots, the fragmentation was approximately first order with respect to intact root number. It was postulated that root breakage in such a high shearing device was due to root‐impeller collisions and the particle size data suggest that roots colliding with the impeller were completely fragmented into debris particles of the order of 0.1 mm in length. IgG release normalized to release by grinding appeared to lag behind the number of roots that had fragmented, suggesting that a process of leakage followed fragmentation in the ultra‐scale down shearing device. Biotechnol. Bioeng. 2014;111: 196–201. © 2013 Wiley Periodicals, Inc.