PEG-mediated Method Research Articles

Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides. In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method. Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%. This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.

An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9.

Protoplasts are commonly used in genetic and breeding research. In this study, the isolation of sorghum protoplasts was optimized and applied to transient gene expression and editing by CRISPR/Cas9. The protoplast was most viable in 0.5 M mannitol, which was the highest of three concentrations after 48- and 72-hours treatments. Using this method we can derive an average of 1.6×106 cells which vary from 5 to 22 nm in size. The average transfection of the protoplasts was 68.5% using the PEG-mediated method. The subcellular assays located Sobic.002G279100-GFP and GFP proteins in the cell compartments as predicted bioinformatically. Two CRISPR/Cas9 plasmids were transfected into sorghum protoplasts to screen for an appropriate sgRNA for gene editing. One plasmid can correctly edit the target region using a single protoplast cell as template DNA. Our results indicated that the protoplast assays as optimized are suitable for transient gene expression and sgRNA screening in CRISPR/Cas9 gene editing procedures.

Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vesca)

Polyethylene glycol (PEG)-mediated transient expression system in plant protoplasts has been widely used in a variety of plants for gene function characterization. However, such a system has not been developed for strawberry. In this study, we report a method for obtaining high quality and high yield protoplasts from strawberry leaves. In addition, we developed an efficient transient gene expression system based on the PEG-mediated method, and the system has been successfully applied to studies of protein expression, protein subcellular localization and protein–protein interaction in strawberry. Furthermore, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable targeted mutagenesis of the FvTCP17 gene in strawberry protoplasts. Taken together, our results demonstrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9-mediated genome editing in strawberry. We report a highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9‐mediated genome editing in strawberry.

RNAi-mediated down-regulation of a melanin polyketide synthase (pks1) gene in the fungus Slafractonia leguminicola.

The fungus Slafractonia leguminicola, the causal agent of blackpatch disease of legumes produces two mycotoxins slaframine and swainsonine, causing slobbers' symptoms and locoism of grazing animals, respectively. The genetics of this important fungus is poorly understood. This work aimed to develop a genetic transformation system and evaluate the efficacy of RNA interference (RNAi) in S. leguminicola. In this study, S. leguminicola was transformed using a PEG-mediated method with a fungal construct that carries a hygromycin resistance cassette. To assess the use of RNAi, a silencing construct pSilentPKS1-AS was constructed which includes inverted repeat transgenes of the polyketide synthase gene (pks1) that is involved in melanin biosynthesis. Transformation of S. leguminicola with the IRT pks1 vector decreased pks1 transcripts levels 82-92% in knockdown mutants when compared with the wild type and was accompanied with a reduction in melanin and swainsonine production. These results demonstrate that RNAi can be a useful tool for studying gene function in S. leguminicola.

Establishment of Genetic Transformation System of Cordyceps militaris using PEG Mediated Method

Establishment of Genetic Transformation System of Cordyceps militaris using PEG Mediated Method

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

Development of a stable genetic system for Chlorella vulgaris—A promising green alga for CO2 biomitigation

Chlorella vulgaris has been proposed as a promising green alga for CO2 biomitigation due to its attractive traits of high growth rate, excellent CO2 fixation ability and broad industrial applications. Nevertheless, the genetic engineering of this alga is still in its infancy. In the present study, a stable and efficient transformation system was established for C. vulgaris CBS 15-2075, a robust strain with great potential for CO2 biomitigation. The antibiotic sensitivity spectrum of this organism was evaluated, and the nptII gene was selected as a dominant selectable marker for genetic transformation. The selectable marker, together with an enhanced green fluorescent protein (EGFP) gene, was delivered into C. vulgaris protoplasts by using a PEG-mediated method, giving a transformation efficiency of 356±30cfu per μg vector DNA. Molecular characterization and live-cell fluorescence microscopy demonstrated that the EGFP gene was stably integrated into C. vulgaris genome and expressed in the cytoplasm of transformed cells. Taken together, we for the first time established a stable and useful genetic toolkit for the industrially important microalga C. vulgaris, which will facilitate to a great extent the future rational genetic manipulation for strain improvement to increase CO2 fixation capacity and may as well provide valuable insights into other Chlorella species. Besides, the success in EGFP expression and live-cell fluorescence detection in C. vulgaris will be useful in molecular and cell biology for protein subcellular localization.

PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts: a valuable tool for signal transduction study in maize

Polyethylene glycol (PEG)-mediated transient gene expression and silencing in protoplasts is widely applied in model plants such as Arabidopsis thaliana and rice. Here, we developed an efficient transient gene expression system based on the PEG-mediated method both in etiolated and green maize mesophyll protoplasts. The results showed that both yellow fluorescent protein encoding gene and glucuronidase encoding gene were efficiently expressed in maize protoplasts. More importantly, double-stranded RNAs (dsRNAs) can also be transfected into maize protoplasts by the PEG-mediated method to specifically silence exogenous and endogenous genes. Our results showed that dsRNA can be used to knockdown both exogenous and endogenous gene expression. Furthermore, bimolecular fluorescence complementation system for the detection of protein–protein interactions in maize protoplasts was developed. We also overexpressed and knockdowned the mitogen-activated protein kinase encoding gene ZmMPK5 to investigate the role of ZmMPK5 in abscisic acid (ABA)-induced antioxidant defense in maize protoplasts. This method here we reported will be valuable for signal transduction study in maize.

The behavior of Bupleurum scorzonerifolium as a parent of somatic hybrid introgressed lines is associated with UV resistance of its chromosomes

Protoplasts of Bupleurum scorzonerifolium irradiated with 380 μW/cm2 UV for 5 min were fused by the PEG-mediated method with untreated protoplasts of Arabidopsis thaliana. The fusion products were cultured in the P5 liquid medium for single hybrid cell clone formation. As a total, 81 independent putative hybrid clones (cell lines) were obtained, and seventeen of them were identified as somatic hybrids by chromosome counting, GISH, RAPD, and SSR analyses. More than 80 B. scorzonerifolium-like green introgressed plants and leaves were regenerated from 49 somatic hybrid cell lines, which contained chromatin and DNA characteristic of A. thaliana. To assess the UV tolerance of both parents with chromatin exclusion and introgression, their protoplasts were UV-irradiated (380 μW/cm2 for 0 and 5 min), and the protoplasts of A. thaliana were more sensitive to UV than those of B. scorzonerifolium as judged by Single Cell Gel Electrophoresis analysis. The possible relationship between UV resistance of B. scorzonerifolium and A. thaliana chromosome elimination and the formation of somatic introgressed hybrid plants is discussed.

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l−1 2,4-D and 0.5 mg l−1 kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l−1 2,4-D and 0.5 mg l−1 kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l−1 IAA and 1.0–3.0 mg l−1 BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l−1 BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.

Antigen-Based Immunofluorescence Analysis of B-Cell Targeting: Advanced Technology for the Generation of Novel Monoclonal Antibodies with High Efficiency and Selectivity

On the basis of immunofluorescence analysis, an entire pathway of B-cell targeting was successfully identified, which can drive selective production of monoclonal antibodies with high efficiency and selectivity. The technique comprises three critical steps, antigen-based preselection of B lymphocytes, formation of antigenselected B lymphocyte and myeloma cell complexes, and selective fusion of B-cell-myeloma cell complexes with electrical pulses. Intriguingly, expression of surface immunoglobulin receptors on B lymphocytes was recognized even after immunization in vitro. The number of the antigen-selected B lymphocytes after in vitro immunization was in fact higher than that obtained after in vivo immunization, suggesting that such shortterm immunization is applicable for B-cell targeting. Immunofluorescence analysis revealed the targeting technique to demonstrate fivefold to tenfold higher efficiency for formation of hybridoma cells than a polyethylene glycol (PEG)-mediated method. This efficiency is in good agreement with production of hybridoma cells secreting desired monoclonal antibodies determined by an enzyme-linked immunosorbent assay (ELISA). The addition of a low concentration of PEG brought about enhanced fusion efficiency and reduced cell damage at even electric intensities as high as 4.0 kV/cm and 5.0 kV/cm. Here we demonstrate that immunofluorescence analysis can successfully clarify an entire pathway of B-cell targeting and provide advantages over the PEG-mediated method. This advanced technology may be applicable for rapid production of monoclonal antibodies based on in vitro immunization.

High UV-tolerance with introgression hybrid formation of Bupleurum scorzonerifolium Willd

The protoplasts of Arabidopsis thaliana (L.) Heynh. treated with 7.5 mM of IOA (iodoacetoamide) were fused with those of Bupleurum scorzonerifolium Willd, using PEG-mediated method. The protoplasts of B. scorzonerifolium were irradiated with ultraviolet light (UV) at an intensity of 380 μW/cm 2 for 30, 60, 90, and 120 s, respectively, before fusion. In cell clones produced in all the fusion combinations, only those from Combination VI (UV 120 s) differentiated to plantlets. The cell lines and plantlets were identified as somatic hybrids by analysis of chromosome size and number, RAPD, 5S rDNA spacer sequence respectively. Genomic in situ hybridization (GISH) revealed that there was a complete set of chromosomes of donor ( B. scorzonerifolium) in all hybrids with a few intact chromosomes and chromosome segment(s) of recipient ( A. thaliana) in the hybrid cell lines from Combinations I–V or only chromosomal segment(s) in the regenerated cell lines from Combination VI. The numbers of chromosomal fragments from A. thaliana translocated or inserted to donor chromosomes increased with UV dose enhanced. The UV-tolerance of B. scorzonerifolium and indirect effect of UV on the exclusion and introgression of receptor ( A. thaliana) chromosomes in the hybrid cells was discussed.

Development of a genetic system in chitinase-producing streptomyces and the application of an allosamidin-insensitive chitinase gene to homologous overexpression.

A transformation system for Streptomyces sp. AJ9463 strain (allosamidin producer) was successfully developed using protoplasts and a PEG-mediated method. To prepare protoplasts, the concentration of glycine and sucrose in YEME medium were optimized to 0.5% (w/v) and 34.0% (w/v), respectively. When the protoplasts of Streptomyces sp. AJ9463 were transformed with pUWL-KS, transformants could be obtained at a high efficiency of 7.0 x 10(4) transformants per microg DNA. To ensure that the transformation system worked properly, we then constructed a constitutive expression vector pYK1, in which the ermE* promoter drives transcription of the allosamidin-insensitive chitinase gene, chiIS. Although no transformant could be obtained by the genetic system using pYK1 isolated from Escherichia coli DH5alpha, pYK1 isolated from the methylase-deficient mutant E. coli SCS110, could be introduced into Streptomyces sp. AJ9463. This indicates that Streptomyces sp. AJ9463 has a methylation-specific restriction system, and that the chiIS and/or ermE* promoter region of pYK1 includes a restriction site of its endonuclease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that pYK1 in Streptomyces sp. AJ9463 started to obviously express ChiIS from 14-h. Moreover, the pYK1-introduced strain gave a five-fold higher chitinase activity than the wild-type, suggesting that this system can be widely applied for the overexpression and gene functional analysis.

Agrobacterium and PEG-mediated transformation of the phytopathogen Venturia inaequalis

We report the development of two new transformation systems, polyethylene glycol (PEG)-mediated transformation of protoplasts and Agrobacterium tumefaciens-mediated transformation of mycelium, for the filamentous ascomycete Venturia inaequalis. New binary vectors have been created for the latter. Although transformation was initially achieved using a PEG-mediated method, this was superseded by the A. tumefaciens-mediated approach. The advantages of the latter include: ease of the protocol, no requirement for protoplasts; higher transformation efficiency; and single-site integration. A comparison between the two transformation methods is presented.