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1.
Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation 总被引:10,自引:0,他引:10
When Agrobacterium was used to transform Nicotiana plumbaginifolia protoplasts and Arabidopsis thaliana roots and seedlings, a large number of plants were found in which not only the T-region defined by the border repeat sequences but the entire binary vector was integrated, as determined by both PCR and Southern analysis techniques. N. plumbaginifolia protoplast co-cultivation experiments yielded 3 out of 5 transformants with collinear sequence past the left border. In Arabidopsis root transformation experiments, 33% (6/18) of the transformants had T-DNA which exceeded the left border repeat. Vacuum infiltration of Arabidopsis seedlings produced even a greater percentage of transformants with sequences outside the left border repeat (62%, 39/63). The long transfer DNA cosegregated with the T-region encoded hygromycin resistance in the T2 progeny eliminating the possibility that long transfer DNA was of extrachromosomal or Agrobacterium origin. The high frequency of long transfer after vacuum infiltration of A. thaliana needs to be considered when analyzing T-DNA tagged mutants. 相似文献
2.
Takayuki Hata Kazuki Mukae Soichrou Satoh Mitsuhiro Matsuo Junichi Obokata 《Plant Biotechnology》2021,38(1):179
The Arabidopsis T87 cell line has been widely used in both basic and biotechnological plant sciences. Agrobacterium-mediated transformation of this cell line was reported to be highly efficient when precultured in Gamborg’s B5 medium for a few days. However, because we could not obtain the expected efficiency in our laboratory, we further examined the preculture conditions of Arabidopsis T87 cells in the Agrobacterium-mediated transformation. As a result, we found that preculture in an excess amount of Murashige and Skoog (MS) macronutrients before cultivation in the B5 medium enhanced the transformation efficiency up to 100-fold, based on the transformed callus number on selective gellan gum plates. In this study, transformants were labeled with green fluorescent protein (GFP), and we found multiple fluorescent spots on individual transgenic calli. Therefore, the actual number of transgenic clones seems much more than that of transgenic calli. In our MS macronutrient-rich culture condition, T87 cells tended to aggregate and formed bigger cell clumps, a change that might be related to the enhancement of transformation efficiency. Based on these results, we report an improved protocol of Agrobacterium-mediated transformation of Arabidopsis T87 cells with high efficiency. 相似文献
3.
S. B. Preuss C.-Z. Jiang H.-K. Baik C. I. Kado A. B. Britt 《Molecular & general genetics : MGG》1999,261(4-5):623-626
Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome. Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although
such factors, presumably DNA repair proteins, are widely presumed to exist. It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis. Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure.
Received: 23 June 1998 / Accepted: 21 February 1999 相似文献
4.
A mini binary vector series for plant transformation 总被引:33,自引:0,他引:33
Xiang Chengbin Han Peng Lutziger Isabelle Wang Kan Oliver David J. 《Plant molecular biology》1999,40(4):711-717
A streamlined mini binary vector was constructed that is less than 1/2 the size of the pBIN19 backbone (3.5 kb). This was accomplished by eliminating over 5 kb of non-T-DNA sequences from the pBIN19 vector. The vector still retains all the essential elements required for a binary vector. These include a RK2 replication origin, the nptIII gene conferring kanamycin resistance in bacteria, both the right and left T-DNA borders, and a multiple cloning site (MCS) in between the T-DNA borders to facilitate cloning. Due to the reduced size, more unique restriction sites are available in the MCS, thus allowing more versatile cloning. Since the traF region was not included, it is not possible to mobilize this binary vector into Agrobacterium by triparental mating. This problem can be easily resolved by direct transformation. The mini binary vector has been demonstrated to successfully transform Arabidopsis plants. Based on this mini binary vector, a series of binary vectors were constructed for plant transformation. 相似文献
5.
【目的】将农杆菌介导的转化应用于重要的工厂化栽培食用菌斑玉蕈中,建立稳定的农杆菌介导的斑玉蕈遗传转化技术。【方法】将构建的双元载体pYN6982转入农杆菌LBA4404菌株中,以斑玉蕈SIEF3133菌株打碎的双核菌丝为受体材料,利用根癌农杆菌介导的转化方法进行斑玉蕈转化试验。【结果】经潮霉素抗性筛选、PCR鉴定以及有丝分裂稳定性试验验证,表明潮霉素磷酸转移酶基因(hph)已经整合到斑玉蕈的基因组中;转基因斑玉蕈菌丝在荧光显微镜下可以观测到绿色荧光,表明增强型绿色荧光蛋白基因(egfp)已经在转基因斑玉蕈菌株中获得了表达;通过PCR检测,随机挑选的8个转基因斑玉蕈菌株中有2个可以扩增出载体转移DNA(T-DNA)边界重复序列外的卡那霉素基因(kan)序列。【结论】获得了稳定遗传和表达的斑玉蕈转基因菌株,建立了农杆菌介导的斑玉蕈遗传转化方法。农杆菌介导的斑玉蕈遗传转化中,存在载体T-DNA边界重复序列之外的DNA序列转移到转基因斑玉蕈中的现象,有待进一步研究。 相似文献
6.
Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pTOK233, which contained the GUS reporter gene and a kanamycin-resistance gene nptII, was employed for optimizing the transformation efficiency evaluated by a GUS gene transient expression level. Eight factors including explant types, explant size and source, the concentration of cytokinin,
inoculation time, pH of inoculation and cocultivation media, bacterial concentration, acetosyringone concentration, and cocultivation
duration were investigated in detail. This optimized protocol was then adopted to obtain transgenic tomato plants resistant
to cucumber mosaic virus (CMV) mediated by Agrobacterium tumefaciens, strain LBA4404, carrying a binary vector pR-ΔGDD containing the kanamy cin-resistance gene and CMV replicase gene with GDD
deletion. The presence of the CMV-RNA2 gene was confirmed by genomic DNA Southern blot analysis in all transformants analyzed. Field spray test showed that the
transgenic tomato plants were resistant to 100 mg/l kanamycin.
Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 280–284.
The text was submitted by the authors in English. 相似文献
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Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis 总被引:2,自引:0,他引:2
Agrobacterium-mediated transient assays for gene function are increasingly being used as alternatives to genetic complementation and stable transformation. However, such assays are variable and not equally successful in different plant species. We analysed a range of genetic and physiological factors affecting transient expression following agroinfiltration, and developed a protocol for efficient and routine transient assays in several plant species. Lettuce exhibited high levels of transient expression and was at least as easy to work with as Nicotiana benthamiana. Transient expression occurred in the majority of cells within the infiltrated tissue and approached 100% in some regions. High levels of transient expression were obtained in some ecotypes of Arabidopsis; however, Arabidopsis remains recalcitrant to routine, genotype-independent transient assays. Transient expression levels often exceeded those observed in stably transformed plants. The laboratory Agrobacterium tumefaciens strain C58C1 was the best strain for use in plant species that did not elicit a necrotic response to A. tumefaciens. A wild A. tumefaciens strain, 1D1246, was identified that provided high levels of transient expression in solanaceous plants without background necrosis, enabling routine transient assays in these species. 相似文献
9.
农杆菌介导的苜蓿次级体细胞胚的遗传转化 总被引:1,自引:0,他引:1
采用农杆菌菌株GV3101感染子叶期苜蓿体细胞胚来研究苜蓿次级体细胞胚的遗传转化方法。农杆菌菌株GV3101双相载体pCAMBIA2301,此双相载体具有gus报告基因和nptⅡ抗卡那霉素筛选基因。感染的子叶期苜蓿体细胞在75 mg/L卡那霉素筛选压下,经过一系列诱导培养,最终获得转基因植株。然后,通过GUS组织化学定位分析来检测转基因植株不同器官中的GUS表达,并进一步通过PCR和Southern杂交确定转基因的稳定整合和转化率。结果表明转基因植株不同器官均有GUS表达,整合的nptⅡ基因的拷贝数是1~4,获得的转基因植株的转化率是65.82%。 相似文献
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We report the construction of a binary vector for Agrobacterium tumefaciens-mediated transformation, pBIN20, which contains a superlinker region located between the left and right Ti border sequences. This vector, derived from pBI121, simplifies the cloning of plant expression cassettes and has been used in our laboratory to create lines of transgenic BY-2 tobacco cells. This new vector contains more than 20 unique restriction sites as well as the nptII selectable marker gene within the Ti-DNA borders. 相似文献
12.
Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression 总被引:23,自引:0,他引:23
Sonication-assisted Agrobacterium-mediated transformation (SAAT) tremendously improves the efficiency of Agrobacterium infection by introducing large numbers of microwounds into the target plant tissue. Using immature cotyledons of soybean
as explants, we evaluated the effects of the following parameters on transient β-glucuronidase (GUS) activity: cultivars, binary vectors, optical density of Agrobacterium during infection, duration of sonication treatment, co-culture conditions, length of explant preculture and addition of acetosyringone
during co-culture. The extent of tissue disruption caused by sonication was also determined. The highest GUS expression was
obtained when immature cotyledons were sonicated for 2 s in the presence of Agrobacterium (0.11 OD600nm) followed by co-cultivation with the abaxial side of the explant in contact with the culture medium for 3 days at 27°C. The
addition of acetosyringone to the co-culture medium enhanced transient expression. No differences were observed when different
cultivars or binary vectors were used. Cotyledons sonicated for 2 s had moderate tissue disruption, while the longer treatments
resulted in more extensive damage.
Received: 1 October 1997 / Revision received: 18 February 1998 / Accepted: 13 March 1998 相似文献
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Nicolau Sbaraini Mariana Vieira Tomazett Augusto Bartz Penteriche Relber Aguiar Gonales Matheus da Silva Camargo Alexandre Melo Bailo Clayton Luiz Borges Augusto Schrank Clia Maria de Almeida Soares Charley Christian Staats 《Genetics and molecular biology》2021,44(3)
Filamentous fungi are the organisms of choice for most industrial biotechnology. Some species can produce a variety of secondary metabolites and enzymes of commercial interest, and the production of valuable molecules has been enhanced through different molecular tools. Methods for genetic manipulation and transformation have been essential for the optimization of these organisms. The genus Simplicillium has attracted increased attention given several potential biotechnological applications. The Simplicillium genus harbors several entomopathogenic species and some isolates have been explored for bioremediation of heavy metal contaminants. Furthermore, the myriad of secondary metabolites isolated from Simplicillium spp. render these organisms as ideal targets for deep exploration and further biotechnological mining possibilities. However, the lack of molecular tools hampered the exploration of this genus. Thus, an Agrobacterium tumefaciens-mediated transformation method was established for Simplicillium subtropicum, employing the far-red fluorescent protein TURBOFP635/Katushka, as a visual marker, and the selection marker SUR gene, that confers resistance to chlorimuron ethyl. Notably, one round of transformation using the established method yielded almost 400 chlorimuron resistant isolates. Furthermore, these transformants displayed mitotic stability for, at least, five generations. We anticipate that this method can be useful for deep molecular exploration and improvement of strains in the Simplicillium genus. 相似文献
15.
Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange 总被引:6,自引:0,他引:6
Transgenic Washington navel orange [Citrus sinensis (L.) Osbeck] plants were obtained using Agrobacterium-mediated transformation of seedling epicotyl tissue. An average of 45% (58 out of 128 segments) of the epicotyl segments
produced shoots expressing the β-glucuronidase (GUS)-intron reporter gene when using Agrobacterium strain C58 C1, compared to 29% (38 out of 128 segments) for EHA101-5 and 0% for LBA4404. Co-culture of 21-day-old Washington
navel epicotyl stem segments gave greater transformation efficiency than co-culture of 35- or 56-day-old stem segments. After
6 weeks, regenerated shoots were micro-grafted in vivo onto seedling rootstocks of Carrizo citrange. Stable integration of
the transgene sequence was confirmed by expression of the plant intron-containing GUS gene, PCR and Southern hybridization.
The apomictic (non-zygotic) state of the transgenic plants was confirmed by isoenzyme and random amplified polymorphic DNA
analyses. More than 50 transgenic plants have been obtained and are growing in the greenhouse.
Received: 14 April 1998 / Revision received: 9 June 1998 / Accepted: 8 July 1998 相似文献
16.
Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as
explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot
regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium
containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot
meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants
and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination
percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with
50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized.
Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 462–467.
The text was submitted by the authors in English. 相似文献
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18.
Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral Fantasy×Phalaenopsis (Baby Hat×Ann Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (GUS) and hygromycin resistance.
The efficiency of transformation was markedly increased by 10 h cocultivation of cell clumps with A. tumefaciens that had been induced with 200 μm acetosyringone, and by inclusion of 500 μm acetosyringone in the cocultivation medium. Hygromycin-resistant cell clusters (0.5–3 mm in diameter) were selected from
the infected cell clumps after 4–6 weeks of culture on agar (8 g/l)-solidified new Dogashima medium (NDM) containing 20 g/l
sucrose, 0.1 mg/l naphthaleneacetic acid, 1.0 mg/l benzyladenine (BA), 50 mg/l hygromycin and 300 mg/l cefotaxime. The cell
clusters proliferated 4 weeks after transfer onto the same medium. To induce callus greening, the carbon source was changed
from sucrose to maltose. The green calli obtained produced protocorm-like bodies (PLBs) after 4 weeks of culture on phytohormone-free
NDM medium. Regeneration of transgenic plantlets was enhanced by incubating PLBs on NDM medium supplemented with 0.1 mg/l
abscisic acid, followed by partial desiccation for 10–30 min. Successful transformation was confirmed by histochemical GUS
assay, PCR analysis and Southern hybridization of transformants. With this transformation system, more than 100 hygromycin-resistant
phalaenopsis plantlets were produced about 7 months following infection of the cell aggregates.
Received: 10 November 1998 / Revision received: 4 June 1999 / Accepted: 22 June 1999 相似文献
19.
缺刻缘绿藻(Myrmecia incisa)系单细胞淡水绿藻,能够大量合成并积累花生四烯酸(arachidonic acid,ArA,20:4ω6),尤其在氮饥饿条件下.基于该绿藻中的延长酶基因序列构建双元表达载体,通过根癌农杆菌(Agrobacterium tumefaciens)介导侵染拟南芥(Arabidopisis thaliana),筛选得到携带MiFAE基因的拟南芥转化株.在转基因第3代(T3)植株中应用PCR扩增目的基因片段和GUS染色,分别在DNA、mRNA和表达水平上均成功地检测到MiFAE基因的存在.GC-MS对不同组织甲酯化的脂肪酸进行检测,结果表明,在转基因的拟南芥营养生长期的叶片中,十六碳三烯酸(hexadecaterienoic acid,C16:3△7,10,13)和α-亚麻酸( α-linolenic acid,C18:3△9,12,15,ALA)在总脂肪酸中的百分含量与对照组相比明显下降,分别由10.5%和41.5%降到1.8%和19.6%.结合GUS染色结果,推测这些减少的产物可能通过外源MiFAE基因的作用,直接参与了蜡质或角质的合成代谢途径. 相似文献
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