scholarly journals A method combining TA cloning and fluorescence screening for rapid acquisition of transgenic seeds

BioTechniques ◽  
2020 ◽  
Vol 68 (5) ◽  
pp. 251-256
Author(s):  
Min Li ◽  
Ling-Yan Jiang ◽  
Qin Liu ◽  
Yuan-Hang Wu ◽  
Guo-Dao Liu ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Selection Process ◽  
Work Conditions ◽  
Expression Vectors ◽  
Process Time ◽  
Screening Process ◽  
Rapid Acquisition ◽  
Growth Defects ◽  
Transgenic Seeds ◽  
Foreign Genes

The establishment of transgenic plants has greatly promoted the progress of plant research. However, traditional selection methods using antibiotics or herbicides may miss any positive transformants with growth defects. Additionally, screening with antibiotics/herbicides requires a huge amount of seeds, sterile work conditions and a large amount of space to germinate plants, making the selection process time- and labor-consuming. In this study, we constructed a novel stable transformation vector, plasmid of OLE1-GFP T-DNA vector (pOGT), which can shorten the steps of cloning foreign genes into expression vectors by using TA cloning. Additionally, selection of transformed seeds with fluorescence overcomes the difficulties of conventional selection with antibiotics/herbicides and simplifies the screening process for transgenic plants.

scholarly journals Construction of CRISPR/Cas9 expression vectors habouring gRNA targeted on SLIAA9 gene of tomato

2020 ◽  
Vol 18 (1) ◽  
pp. 147-156
Author(s):  
Bui Manh Minh ◽  
Ha Hong Hanh ◽  
Le Thi Thu Hien ◽  
Huynh Thi Thu Hue
Keyword(s):  
Transgenic Plants ◽  
Genome Editing ◽  
Tomato Fruit ◽  
Expression System ◽  
Secondary Compounds ◽  
Expression Vectors ◽  
Tomato Plants ◽  
Normal Morphology ◽  
Transgenic Tomato Plants ◽  
Pcr Test

Tomato (Solanum lycopersicum) is a nutritious fruit containing many secondary compounds with health benefits. The formation of tomato fruit through fertilization is controlled by auxin through Aux/IAA9 and ARF8 proteins. The mutated SlIAA9 gene leads to the parthenocarpic development of fruit or seedless tomato fruit. Nowadays, the CRISPR/Cas9 genome editing system is becoming increasingly popular in modifying desired genes on plant objects. In this study, gRNAs which target on tomato SlIAA9 gene were designed and inserted into CRISPR/Cas9 vectors. In addition, two strains of A. tumefaciens harboring pRGEB31-IAA9G2 and pRGEB32-IAA9G2 vectors carrying CRISPR/Cas9 expression system towards SlIAA9 gene in tomato were successfully created. The strain of A. tumefaciens harboring pRGEB31- IAA9G2 plasmid was used to develop transgenic tomato plants from Micro-Tom variety. PCR test showed that 5/14 plants had the presence of Cas9 gene in T0 plants. The transgenic plants have a normal morphology in comparation with the controls. The evaluation of mutant efficiency, type, and stability of mutations on the SlIAA9 will be conducted on next-generation plants when the mutations are stable and segregated into descendents.

Ectopic expression of a R2R3 MYB transcription factor of dove tree (Davidia involucrata) aggravates seed abortion in Arabidopsis thaliana

2020 ◽  
Vol 47 (5) ◽  
pp. 454
Author(s):  
Jian Li ◽  
Tian Chen ◽  
Fengzhen Huang ◽  
Penghui Dai ◽  
Fuxiang Cao ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Transcription Factor ◽  
Transgenic Plants ◽  
Ectopic Expression ◽  
Myb Transcription Factor ◽  
Seed Abortion ◽  
Transgenic Seeds ◽  
Genes Encoding ◽  
Colour Changes ◽  
R2r3 Myb

Serious seed abortion of dove tree (Davidia involucrate Baill.) is one of the critical factors leading to the low fecundity of this species. Seed abortion is a complicated process and various factors have been verified to synergistically determine the fate of seeds. To reveal the mechanism of seed abortion in D. involucrata, we performed transcriptome analysis in normal and abortive seeds of D. involucrata. According to the transcriptome data, we noticed that most of the genes encoding a MYB transcription factor were predominantly expressed in abortive seeds. Among these, a gene named DiMYB1 was selected and its function was validated in this study. Overexpression of DiMYB1 resulted in obviously reduced viability of transgenic seeds and seedlings, and caused a significantly higher seed abortion rate. The vegetative growth of transgenic plants was hindered, resulting in an earlier flowering time. In addition, colour changes occurred in transgenic plants. Some transgenic sprouts, stems and pods appeared purple instead of green in colour. Our finding demonstrated that DiMYB1 participates in multiple plant developmental processes, especially in seed development in Arabidopsis thaliana (L.) Heynh., which indicated the similar role of this gene in D. involucrata.

Readiness Criteria: Indonesias’ New Initiative to Ensure Sustainable Development Program

2015 ◽  
Vol 776 ◽  
pp. 101-107
Author(s):  
Alit Merthayasa
Keyword(s):  
Sustainable Development ◽  
Local Governments ◽  
Selection Process ◽  
National Development ◽  
Development Program ◽  
Development Planning ◽  
Assessment Tools ◽  
Funding Agency ◽  
Screening Process ◽  
Donor Funding

Government of Indonesia through Ministry of National Development Planning/Head of BAPPENAS, recently launched a new initiative in regard to Sustainable Development Program/project criteria called Readiness Criteria. The purpose of the criteria is to select proposed development program / project submitted by local governments as well as donor/funding agency especially related to grant or loan funds, through assessment or screening process. The main criteria consist of: relevance, effectiveness, efficiency, sustainability and impact criteria. The screening process implemented using design and monitoring framework (DMF), which is introduced and developed by ADB funded project’s in Indonesia. Decision of the selection process was made based on project assessment tools (PAT). Nowadays, readiness criteria will be implemented during pre-design phase or during proposal submission stages under the planning and budgeting period as stated by Indonesia Development Planning Board/BAPPENAS.

scholarly journals Development of Opsonic Mouse Monoclonal Antibodies against Multidrug-Resistant Enterococci

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Ermioni Kalfopoulou ◽  
Diana Laverde ◽  
Karmela Miklic ◽  
Felipe Romero-Saavedra ◽  
Suzana Malic ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Capsular Polysaccharide ◽  
Selection Process ◽  
Multidrug Resistant ◽  
Expression Vectors ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Strains ◽  
Variable Regions ◽  
Content Type

ABSTRACTMultidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide ofEnterococcus faecalis, and the secreted antigen A (SagA), an immunogenic protein fromEnterococcus faecium, are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing againstE. faecalistype 2, and SagA.01 showed 40% killing againstE. faecium11231/6. In addition, both MAbs showed cross-reactivity toward otherE. faecalisandE. faeciumstrains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.

scholarly journals Coronaviruses as Vectors: Position Dependence of Foreign Gene Expression

2003 ◽  
Vol 77 (21) ◽  
pp. 11312-11323 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Linda van Genne ◽  
Jeroen N. Stoop ◽  
Haukeline Volders ◽  
Peter J. M. Rottier
Keyword(s):  
Gene Expression ◽  
Expression Vectors ◽  
Luciferase Expression ◽  
Foreign Gene ◽  
Regulatory Sequences ◽  
Expression Levels ◽  
Genomic Position ◽  
Position Dependence ◽  
Foreign Genes

ABSTRACT Coronaviruses are the enveloped, positive-stranded RNA viruses with the largest RNA genomes known. Several features make these viruses attractive as vaccine and therapeutic vectors: (i) deletion of their nonessential genes is strongly attenuating; (ii) the genetic space thus created allows insertion of foreign information; and (iii) their tropism can be modified by manipulation of the viral spike. We studied here their ability to serve as expression vectors by inserting two different foreign genes and evaluating systematically the genomic position dependence of their expression, using a murine coronavirus as a model. Renilla and firefly luciferase expression cassettes, each provided with viral transcription regulatory sequences (TRSs), were inserted at several genomic positions, both independently in different viruses and combined within one viral genome. Recombinant viruses were generated by using a convenient method based on targeted recombination and host cell switching. In all cases high expression levels of the foreign genes were observed without severe effects on viral replication in vitro. The expression of the inserted gene appeared to be dependent on its genomic position, as well as on the identity of the gene. Expression levels increased when the luciferase gene was inserted closer to the 3′ end of the genome. The foreign gene insertions generally reduced the expression of upstream viral genes. The results are consistent with coronavirus transcription models in which the transcription from upstream TRSs is attenuated by downstream TRSs. Altogether, our observations clearly demonstrate the potential of coronaviruses as (multivalent) expression vectors.

Construction of Two Vectors for a Bypass of Monocotyledon Plants Photorespiration

2011 ◽  
Vol 340 ◽  
pp. 351-356
Author(s):  
Xue Liang Bai ◽  
Dan Wang ◽  
Ning Ning Liu ◽  
Li Jing Wei ◽  
Ye Rong Zhu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Transgenic Plants ◽  
Binary Vector ◽  
Transit Peptide ◽  
Expression Vectors ◽  
Chloroplast Transit Peptide ◽  
E Coli ◽  
Plant Expression ◽  
Glycolate Dehydrogenase ◽  
Save Energy

In order to modify the photorespiration of monocotyledonous crops, we aimed to construct vectors that will be used to introduce a bypass to the native photorespiration pathway. Firstly, we cloned the encoding sequences of glyoxylate carboligase (GCL) and tartronic semialdehyde reductase (TSR) fromE. coli, glycolate dehydrogenase (GDH) fromArabidopsis thalianaand chloroplast transit peptide (cTP) from rice. Then we constructed a universal vector pEXP harboring the encoding sequence of cTP for targeting a protein into chloroplast. By insertion of these three encoding sequences into the universal vector pEXP, we obtained the expression cassettes for GCL, TSR and GDH, respectively. Finally, we inserted the cassettes for GCL and TSR in tandem into the binary vector pCAMBIA 1301, and for GDH into another binary vector, pPGN, to obtain our plant expression vectors pCAMBIA 1301-TG and pPGN-GDH, respectively. These two expression vectors possess different selection resistance and can be used to transform monocots together, to introduce the bypass pathway of photorespiration. By this way, the transgenic plants can recycle glycolate, the by-product of photosynthesis in C3plants, within the chloroplast, simultaneously, save energy and avoid the loss of ammonia, which will contribute to improved growth.

The Phytoremediation of Radioactively Contaminated Land: A Feasible Approach or Just Bananas?

2013 ◽  
Author(s):  
Victoria A. Nesbitt
Keyword(s):  
Plant Species ◽  
Selection Process ◽  
Extended Period ◽  
Terrestrial Ecosystems ◽  
Nuclear Industry ◽  
Nuclear Facilities ◽  
Screening Process ◽  
Preliminary Screening ◽  
A Site ◽  
Ground Contamination

Soil is an essential component of all terrestrial ecosystems and is under increasing threat from human activity. Techniques available for removing radioactive contamination from soil and aquatic substrates are limited and often costly to implement; particularly over large areas. Frequently, bulk soil removal, with its attendant consequences, is a significant component of the majority of contamination incidents. Alternative techniques capable of removing contamination or exposure pathways without damaging or removing the soil are therefore of significant interest. An increasing number of old nuclear facilities are entering ‘care and maintenance’, with significant ground contamination issues. Phytoremediation — the use of plants’ natural metabolic processes to remediate contaminated sites is one possible solution. Its key mechanisms include phytoextraction and phytostabilisation. These are analogues of existing remedial techniques. Further, phytoremediation can improve soil quality and stability and restore functionality. Information on the application of phytoremediation in the nuclear industry is widely distributed over an extended period of time and sources. It is therefore difficult to quickly and effectively identify which plants would be most suitable for phytoremediation on a site by site basis. In response, a phytoremediation tool has been developed to address this issue. Existing research and case studies were reviewed to understand the mechanisms of phytoremediation, its effectiveness and the benefits and limitations of implementation. The potential for cost recovery from a phytoremediation system is also briefly considered. An overview of this information is provided here. From this data, a set of matrices was developed to guide potential users through the plant selection process. The matrices take the user through a preliminary screening process to determine whether the contamination present at their site is amenable to phytoremediation, and to give a rough indication as to what plants might be suitable. The second two allow the user to target specific plant species that would be most likely to successfully establish based on prevailing site conditions. The outcome of this study is a phytoremediation tool that can facilitate the development of phytoremediation projects, avoiding the need for in-depth research to identify optimal plant species on a case-by-case basis.

scholarly journals GENETIC TRANSFORMATION AND REGENERATION OF TRANSGENIC SWEETPOTATO PLANTS.

HortScience ◽  
1995 ◽  
Vol 30 (3) ◽  
pp. 435f-435 ◽  
Author(s):  
Marceline Egnin ◽  
C.S. Prakash
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Binary Vector ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Ms Medium ◽  
Petiole Explants ◽  
Efficient Delivery ◽  
Foreign Genes

This study aimed to optimize factors for the efficient delivery of foreign genes into sweetpotato using Agrobacterium tumefaciens and develop transgenic plants. Disarmed Agrobacterium C58 carrying a binary vector pBI 121C2H with gusA, nptll, and the nutritional protein asp-l genes was used to cocultivate (4 days) petiole explants of the sweetpotato genotype P1318846-3. Pre-incubation of petioles for 3 days on MS medium with 2,4-D (0.2 mg·liter–1) before infection resulted in higher transformation. Putative transgenic shoots were obtained by transfer of petioles to MS medium with TDZ (0.2 mg·liter–1) and kanamycin (80 to 140 mg·liter–1). The PCR amplification of gusA, nptll, and asp-1 genes in the 37 putative transgenic shoots showed that six plants contained the three genes. However, none of these plants showed histochemical expression of the gusA gene. The introduced gene may have been methylated resulting in the lack of its expression. DNA blot hybridization studies are underway to verify the presence and integration of the transgenes.

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