Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants

We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover, we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity, but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.     

Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers

Cereal crops have been the primary targets for improvement by genetic transformation because of their worldwide importance for human consumption. For a long time, many of these important cereals were difficult to genetically engineer, mainly as a result of their inherent limitations associated with the resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. The delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique. However, there are still serious handicaps with Agrobacterium -mediated transformation of other major cereals. In this paper, we review the pioneering efforts, existing problems and future prospects of Agrobacterium -mediated genetic transformation of major cereal crops, such as rice, maize, wheat, barley, sorghum and sugarcane.     

Transgenic Populus tremula: a step-by-step protocol for its Agrobacterium-mediated transformation

In recent years, Populus species have acquired an important place in basic and applied research of woody plants. The practical role of Populus species in world forestry and their importance to research as a woody-plant model have led to increasing interest in tissue-culture and molecular techniques, as well as the development of transformation procedures for this genus. A simple technical procedure is described here step-by-step, for the first time, as a routine method for transforming Populus tremula using a disarmed Agrobacterium tumefaciens hypervirulent strain. The procedure begins with the inoculation of stem explants with bacterial suspension, followed by a short period of co-cultivation on a highly regenerative medium. Transformed shoots are selected on regeneration medium containing antibiotics and the presence of the inserted target genes is checked using a rapid and efficient PCR test. Selected shoots are transferred to a rooting medium, under the same selection pressure, and propagated via stem cuttings. Selected plants can be hardened and transferred to the green-house within 4 months of inoculation. The method has proven efficient for several gene constructs, selection on Kan or Hyg, and three different Agrobacterium strains.     

根癌农杆菌介导的香蕉遗传转化研究进展

香蕉的栽培品种绝大多数是三倍体, 常规育种难度大, 工作周期长, 难以采用传统的育种方法进行遗传改良, 因此转基因技术成为改良香蕉种质的有效方法。根癌农杆菌介导的香蕉的遗传转化从20世纪90年代起取得的了较大的进步, 但仍然存在着很多问题。文章分析了影响农杆菌介导香蕉转化的几个重要环节, 并对其研究现状、存在的问题及应用前景作简要概述。     

根癌农杆菌介导转化番茄的影响因素

综述影响根癌农杆菌介导番茄转化效率的因素,包括根癌农杆菌菌株类型、Vir基因的活化、选择标记基因、植物基因型、外植体类型、培养基中是否附加植物激素和抑菌抗生素、菌液浓度、侵染时间长短,是否预培养和共培养天数等;同时不同的培养方式也是影响番茄转化效率的主要因素,包括液体培养法、农杆菌介导的floral-dip转化法、超声波辅助农杆菌介导法、农杆菌介导与基因枪轰击结合法等.     

根癌农杆菌介导蓝猪耳转化的影响因素研究

研究了根癌农杆菌介导蓝猪耳转化的影响因子。结果表明,以蓝色花类型蓝猪耳5~6周的叶片为外植体,在OD₆₀₀为0.5~0.6的活化菌液中浸染5~10min,暗培养4d后,在愈伤组织诱导培养基(MS+BA 1.0mg/L+2,4-D 0.1mg/L)上生长14d,获得抗性愈伤组织;经芽诱导培养基(1/2MS+BA 1.0mg/L+NAA 0.1mg/L)培养28d得到抗性芽;生根培养基(1/2MS)上培养14d得到抗性植株。经PCR检测证实外源基因已整合到蓝猪耳基因组中,转化率达13%~14%。Cef和Hyg浓度对转化影响较大,转化的不同阶段其适宜浓度不同。     

pBIN20: An Improved Binary Vector for shape Agrobacterium-mediated Transformation

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.     

SAAT: sonication-assisted Agrobacterium-mediated transformation

Plant transformation via Agrobacterium can be limited by both host specificity and the inability of Agrobacterium to reach the proper cells in the target tissue. Described here is a new and efficient Agrobacterium-based transformation technology that overcomes these barriers and enhances DNA transfer in such diverse plant groups as dicots, monocots, and gymnosperms. This new technology, called sonication-assisted Agrobacterium-mediated transformation (SAAT), involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium. Scanning electron and light microscopy reveal that SAAT treatment produces small and uniform fissures and channels throughout the tissue allowing the Agrobacterium easy access to internal plant tissues. Unlike other transformation methods, this system has the potential to transform meristematic tissue buried under several cell layers. SAAT increases transient transformation efficiency in several different plant tissues including leaf tissue, immature cotyledons, somatic and zygotic embryos, roots, stems, shoot apices, embryogenic suspension cells and whole seedlings. A 100- to 1400-fold increase in transient - glucuronid ase expression has been demonstrated in various tissues of soybean, Ohio buckeye, cowpea, white spruce, wheat and maize. Stable transformation of both soybean and Ohio buckeye has been obtained using SAAT of embryogenic suspension culture tissues. For soybean, SAAT treatment was necessary to obtain stable transformation with this tissue     

农杆菌介导的斑玉蕈遗传转化

【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。     

毛白杨高效转化系统的研究(英文)

毛白杨是一种具有多种优良特性、用途广泛并有重要经济价值的杨属树种,又是农杆菌的天然寄主植物。因此,毛白杨一直是进行木本植物组培及遗传转化研究的重要对象。本研究通过比较培养基组成(Table 1)、外植体类型(Table 2)、农杆菌侵染浓度以及预培养(Table 3&4, Fig.1)和共培养条件(Table 5)等影响农杆菌转化及植株再生效率的诸多重要条件后,建立了毛白杨的农杆菌高效转化系统。实验选用毛白杨叶片为外植体,在MS + 4.0 mg/L 2,4-D + 1.0 mg/L 6-BA培养基上预培养2 d后制成叶盘,并做戳伤处理,再与5×108 Cell/mL浓度的农杆菌新鲜菌液共培养2 d,然后以MS + 4.0 mg/L 2,4-D + 1.0 mg/L 6-BA附加50 mg/L Km和1 000 mg/L AP为筛选培养基(Table 6, Fig.4) ,获得最佳转化效果。2－3周后,在叶盘周围长出许多Km抗性愈伤组织,转入分化培养基分化后,再转入生根培养基3－4周,获得完整植株(Fig.3)。经PCR扩增实验鉴定,转化愈伤组织的阳性率达到83.8％(Fig.2)。多次重复实验结果表明,该转化系统与以往的文献报道相比,其遗传转化率大大提高。抗性愈伤组织的平均转化率高达56.2%,最终得到的再生转化植株的比率达19.9%。其中,毛白杨叶盘经戳伤处理,其转化率可提高至20~40% (Table 7)。利用此转化系统     

Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre

The Cre/lox system was used to obtain targeted integration of an Agrobacterium T-DNA at a lox site in the genome of Arabidopsis thaliana. Site-specific recombinants, and not random events, were preferentially selected by activation of a silent lox-neomycin phosphotransferase (nptII) target gene. To analyse the effectiveness of Agrobacterium-mediated transfer we used T-DNA vectors harbouring a single lox sequence (this vector had to circularize at the T-DNA left- and right-border sequences prior to site-specific integration) or two lox sequences (this vector allowed circularization at the lox sequences within the T-DNA either prior to or after random integration, followed by targeting of the circularized vector), respectively. Furthermore, to control the reversibility of the integration reaction, Cre recombinase was provided transiently by using a cotransformation approach. One precise stable integrant was found amongst the recombinant calli obtained after transformation with a double-lox T-DNA vector. The results indicate that Agrobacterium-mediated transformation can be used as a tool to obtain site-specific integration.     

Multiple pathways for Cre/lox-mediated recombination in plastids

Plastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to approximately 10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker-free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre-mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome 'hot spot' of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans-splicing events in higher plant plastids.     

Cre-mediated site-specific gene integration for consistent transgene expression in rice

To minimize expression variability amongst transgenic lines, we have utilized the strategy of Cre/lox-mediated site-specific gene integration. This method allows the precise integration of a transgene in a lox site previously placed in the genome. Using the biolistic method for DNA delivery, we have generated several site-specific integrant lines, derived from three different target lines. About 80% of the selected lines contain precise integration of the gusA reporter gene and fall into two categories: single-copy (SC) lines that contain site-specific integration without additional random integrations, and multicopy (MC) lines that contain random integrations in addition to the site-specific integration. The expression of the gusA gene was studied in callus cells and regenerated plants. The isogenic SC lines displayed significantly lower expression variation, whereas much higher expression variation was observed in MC lines. Furthermore, stable inheritance of the gusA gene was observed in T1 plants derived from a subset of SC lines. This demonstrates that consistent gene expression can be obtained in rice by Cre-mediated site-specific integration.     

A Simple Method for Identifying Plant/T-DNA Junction Sequences Resulting from Agrobacterium-mediated DNA Transformation

Genomic integration of transferred T-DNA is traditionally analyzed by Southern hybridization; however, these analyses often do not provide sufficient information pertaining to the transformation event. Analysis of the junction sequences spanning the region between the T-DNA borders and plant genomic DNA, give a clear demonstration of genomic integration. The procedures available for border junction analysis can be problematic, therefore a simplified method was developed for plants transformed by Agrobacterium tumefaciens harboring the binary vector with pBI121 backbone.     

根癌农杆菌对巴戟天遗传转化的影响因素

以巴戟天带节茎为材料,研究了根癌农杆菌对巴戟天遗传转化的影响因素。结果表明:外植体感染前先进行2 d预培养,对转化有一定促进作用;外植体与农杆菌共培养时间以3 d为宜;乙酰丁香酮能提高转化效率,抗性芽分化率可达18.0％;外植体与农杆菌共培养后延迟4 d选择,抗性芽分化率有所提高;硝酸银能抑制外植体表面农杆菌的生长,提高GUS阳性芽的比例,硝酸银浓度2 mg/L时,GUS阳性芽比例最高(42.9％)。     

影响农杆菌介导的植物转基因的因素

农杆菌介导法是植物转基因研究中应用最多的方法,具有转化成本低、效率高、可转移较长基因片段等特点.多种因素影响此遗传转化过程中的转化率,包括外植体类型、植物基因型、外植体的处理、菌株和质粒载体类型、预培养以及培养基成份等方面.     

农杆菌介导的大豆高频遗传转化

大豆(Glycine max(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题.为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2 mg/L 6-BA和5 mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01 mg/L TDZ和2mg/L glufosinate的液体培养基中筛选,并每周更换一次培养液.得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1～2个基因拷贝数.该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论.     

农杆菌介导的大豆高频遗传转化

大豆(Glycinemax(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题。为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2mg/L6-BA和5mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01mg/LTDZ和2mg/Lglufosinate的液体培养基中筛选,并每周更换一次培养液。得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1~2个基因拷贝数。该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论。     

Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.     

Transgene expression produced by biolistic-mediated, site-specific gene integration is consistently inherited by the subsequent generations

The efficient production of stable transgenic plants is important for both crop improvement and functional genomics. Site-specific integration of foreign genes into a designated genomic position is an attractive tool for minimizing expression variability between transgenic lines. Here, we studied the utility of a Cre-mediated, site-specific integration approach, facilitated by particle bombardment, for streamlining the production of stable transgenic plants, using rice as a model species. Using this method, we generated 18 different transgenic lines containing a precise integration of a single copy of beta-glucuronidase gene (gusA) into a designated genomic location. Eleven of these lines contained no illegitimate integration in the background (single-copy lines), and seven contained illegitimate integrations in addition to the site-specific integration (multicopy lines). We monitored gusA expression in these lines up to three to four successive generations. Each of the single-copy lines expressed the gusA gene at consistent levels and nearly doubled the expression level in the homozygous state. In contrast, multicopy lines displayed expression variation and gene silencing. In about half of the multicopy lines, however, expression of the site-specific integration locus could be reactivated and stabilized on segregation of the illegitimate integrations, whereas, in the remaining half, expression could not be restored, as they contained genetically linked illegitimate integrations. This study demonstrates that biolistic-mediated, site-specific gene integration is an efficient and reliable tool for streamlining the production of stable transgenic plants.