Construction of protoplast genetic transformation system for Mycena--symbiont of Gastrodia elata / 中国中药杂志

Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.

Current Technologies and Related Issues for Mushroom Transformation

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae / 生物工程学报

To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.

Breeding of high-producing LI-F lipopeptide Paenibacillus polymyxa by protoplast fusion and differential expression analysis of fusion strains / 生物工程学报

Auxotrophic strains of N1-37 (Phe-) and N2-27 (His-), screened from mutations of Paenibacillus polymyxa JSa-9 previously, were used as the parent strains to screen high-producing LI-F antibacterial lipopeptide fusion strain through protoplast fusion with polyethylene glycol as a promote agent. Fusion strain F5-15 was obtained. Then the product of LI-F antibacterial lipopeptide was quantified by HPLC, and the difference of expression of the key genes of lipopeptide synthase between wild strain JSa-9 and the fusion strain was analyzed by real-time PCR. LI-F antibacterial lipopeptide yield of the fusion strain F5-15 was 3.1-fold of the original strain JSa9's, and the expression levels of the target genes were 10.48, 2.48, 2.1 and 11.8 fold of the initial strain JSa-9, respectively.

Protoplasts isolation, purification and plant regeneration of Pinellia cordata / 中国中药杂志

The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.

Preparation and vitality detection of protoplast in Salvia miltiorrhiza Bunge / 生物工程学报

We prepared protoplasts from Salvia miltiorrhiza Bunge suspension culture cells. Then, the protoplasts' vitality and functions were tested by fluorescein diacetate staining method and Fluo-3/AM flourescent probe. The optimal condition of protoplast isolation was Cellulase R-10 1.5%, Pectinase Y-23 0.3%, Macerozyme R-10 0.5%, 40 r/min 12 h, 600 r/min 5 min, and the protoplasts yield was 1.1x10(6) cells/g FW, the vitality was more than 95% by using fluorescein diacetate staining method. It has been confirmed that calcium fluorescent probe Fluo-3/AM can be successfully loaded into protoplasts.

Efficient transient expression to analyze miRNA targets in rice protoplasts / 生物工程学报

Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice.

Antifungal indole and pyrrolidine-2,4-dione derivative peptidomimetic lead design based on in silico study of bioactive peptide families

The rise of opportunistic fungal infections highlights the need for development of new antimicrobial agents. Antimicrobial Peptides [AMPs] and Antifungal Peptides [AFPs] are among the agents with minimal resistance being developed against them, therefore they can be used as structural templates for design of new antimicrobial agents. In the present study four antifungal peptidomimetic structures named C[1] to C[4] were designed based on plant defensin of Pisum sativum. Minimum inhibitory concentrations [MICs] for these structures were determined against Aspergillus niger N402, Candida albicans ATCC 10231, and Saccharomyces cerevisiae PTCC 5052. C[1] and C[2] showed more potent antifungal activity against these fungal strains compared to C[3] and C[4]. The structure C[2] demonstrated a potent antifungal activity among them and could be used as a template for future study on antifungal peptidomemetics design. Sequences alignments led to identifying antifungal decapeptide [KTCENLADTY] named KTC-Y, which its MIC was determined on fungal protoplast showing 25 [micro g/ml] against Aspergillus fumigatus Af293. The present approach to reach the antifungal molecules seems to be a powerful approach in design of bioactive agents based on AMP mimetic identification

Optimized condition for protoplast isolation from maize, wheat and rice leaves / 生物工程学报

Maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) are three staple crops and accordingly it is very meaningful to optimize the condition of their protoplasts isolation. The concentration of the enzyme, the time of isolation and centrifugal force in protoplast isolation were investigated to find their effects on protoplast yield and viability using leaves of maize (Zong 3), wheat (Chinese Spring) and rice (Nipponbare). The results show that the concentration of the enzyme and the time of isolation affected the protoplast yield significantly. Although the yield of protoplast was increased with high concentration of enzyme and long incubated time, it led to too much cells breakdown. The orthogonal experimental design results show that the best condition of maize protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 7 h, 100 x g 2 min and the protoplasts yield was 7x106 cells/g fresh weight (FW); the best condition of wheat protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 5 h, 100 x g 2 min and the protoplasts yield was 6 x 10(6) cells/g FW; the best condition of rice protoplast isolation was Cellulase R-10 2.0%, Macerozyme R-10 0.7%, 50 r/min 7 h, 1 000 x g 2 min and the protoplasts yield was 6x10(6) cells/g FW. The vitalities were more than 90% using fluorescein diacetate staining method. 50%-80% transformation efficiency was obtained when protoplasts were transformed by green fluorescent protein using PEG-Ca2+ method.

Production and characterization of a somatic hybrid of Chinese cabbage and cabbage / 生物工程学报

In order to broaden Chinese cabbage gene pool, we conducted interspecific somatic hybridization between Chinese cabbage (Brassica campestris, 2n=20, AA) and Cabbage (B. oleracea, 2n=18, CC). Protoplasts were isolated from 10-day-old cotyledons and hypocotyls of young seedlings, and fused by 40% polyethylene glycol (PEG). Fused cells were cultured in modified K8p liquid medium supplemented with some plant growth regulators. Fusion products were characterized by their morphological, cytological and molecular biological traits. The results showed that, a total of 35 regenerated green plants were obtained from 320 calli, the plant regeneration frequency was 10.94%, and eleven of which were survived in greenhouse. All regenerants were true hybrids as confirmed by randomly amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis. Ploidy levels of hybrid plants were determined by chromosome counting and flow cytometry. The sum of the chromosome number (2n = 38) from the two fusion patents were found in 36.4% of regeneratns; another 36.4% had chromosomes range to 58-60; 27.2% had more chromosomes ranges to 70-76. All regenerated plants produced normal flowers. We investigated the pollen fertility and seed set after self-pollination and backcrossing with the parental species. For hybrids with chromosomes more than 38 it was possible to obtain some seeds when they after self-pollination. Within the group of hybrids with 38 chromosomes, seed set were very variable, only 0.11 seeds per pod by self-pollination, 0.23-0.76 by open-pollination, 0.02-0.04 by backcrossing with Chinese cabbage. Progeny lines obtained by self-pollination had larger leaves and leaf shapes intermediate of the parental species. Pollen fertility was gradually recovered in the first and second progenies. The backcrossing progeny lines, as a whole, exhibited morphologies were similar to Chinese cabbage. Morphological variations were observed among the somatic hybrids and their progenies.

Produção e regeneração de protoplastos de Colletotrichum gloeosporioides em diferentes condições de cultivo / Production and regeneration of protoplasts from Colletotrichum gloeosporioides in different conditions

A obtenção de protoplastos de fungos, utilizando-se enzimas degradadoras de parede celular, tem sido o método mais utilizado em processos de transformação genética. Foram testados dois tipos de estruturas fúngicas (micélio e conídios), diferentes concentrações enzimáticas (5, 10, 20 mg), estabilizadores osmóticos (NaCl 0,7 mol.L-1 pH 5,7; (NH4)2SO4 1,2 mol.L-1 pH 5,8; KCl 0,7 mol.L-1 pH 5,8; MgSO4 0,7 mol.L-1 pH 5,5; Sacarose 0,5 mol.L-1 pH 5,7; SorbitoL 0,6 mol.L-1 pH 5,7) e seis tempos de exposição dos protoplastos ao sistema lítico, para estabelecer condições otimizadas de obtenção e regeneração de protoplastos de Colletotrichum gloeosporioides, agente relacionado a mancha manteigosa em cafeeiros. Protoplastos de C. gloeosporiodes foram obtidos em maior quantidade quando o micélio foi exposto durante 4 horas, com 10 mg.mL-1 de Lysing Enzime em KCl 0,7 mol.L-1 que se apresentou como melhor estabilizador osmótico, com frequência de regeneração de 11,64%.

The isolation and regeneration of protoplasts from fungal cells, using several cell wall degrading enzymes, has been the most common method to prepare competent cells for genetic studies of filamentous fungi. In this work two types of fungal structures, different enzyme concentrations (5, 10, 20 mg), osmotic stabilizers (NaCl 0,7 mol.L-1 pH 5,7; (NH4)2SO4 1,2 mol.L-1 pH 5,8; KCl 0,7 mol.L-1 pH 5,8; MgSO4 0,7 mol.L-1 pH 5,5; Sacarose 0,5 mol.L-1 pH 5,7; Sorbitol 0,6 mol.L-1 pH 5,7), and six exposure times to establish optimum conditions of isolation and regeneration of protoplasts of Colletotrichum gloeosporioides, agent of blister spot on coffee, were tested. Protoplast of C. gloeosporioides were obtained in greater quantities when the mycelium was exposed for 5 hours with 15mg.mL-1 of Lysing Enzyme in 0.7 mol.L -1 of KCl as osmotic stabilizer, presenting a regeneration rate of 11.64%.

Morphological, cytological, and molecular characterization of hybrids and their progenies derived from the somatic hybridization of Brassica campestris and Brassica oleracea / 生物工程学报

In order to produce interspecific somatic hybrids between Brassica campestris (2n = 20, AA) and Brassica oleracea (2n = 18, CC), we isolated protoplasts from cotyledons and hypocotyls of young seedlings, and fused by 40% polyethylene glycol (PEG). Fused cells were cultured in modified K8p liquid medium supplemented with 0.2 mg/L 2,4-dichorophenoxyacetic acid (2,4-D) +0.5 mg/L 6-benzylaminopurine (6-BA)+0.1 mg/L naphthaleneacetic acid (NAA)+ 0.1 mg/L Kinetin (Kin), 0.3 mol/L sucrose and 0.3 mol/L glucose were used as osmoticum. At the eight-to ten-cell stage, divided cells were transferred to Kao's basal medium supplemented with 0.3 mol/L sucrose as carbon source and 0.1% agarose, 2 mg/L 6-BA+ 2 mg/L Zeatin (ZEA)+1 mg/L NAA+ 0.5 mg/L Kin for callus induction. After 35 days, when small calli reached 2-3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/L Zeatin (ZEA) and 2 mg/L indole-3-acetic acid (IAA). After the length of the shoots reached 1-2 cm, the shoots were transferred to 1/2 MS+0.2 mg/L NAA for root induction. Morphological, cytological and molecular biological analysis methods were used for identification of somatic hybrids. The results showed that, the first cell division occurred during 2-7 days of culture. Five weeks after culture initiation, the plating efficiency attained 0.66%. Finally, the shoot regeneration frequency was 3.7%. A total of eleven regenerated plants were obtained and verified as somatic hybrids by morphological observation and flow cytometry. Cytological studies showed that all tested plants had a chromosome number of 38, the sum of both parents. Hybridity was also confirmed by randomly amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis, indicating that these regenerated plants were all true hybrids of B. campestris and B. oleracea. All amphidiploid somatic hybrids showed low pollen fertility. Pollen fertility was gradually recovered in the first and second progenies.

Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis

Despite the recent progress of transient gene expression systems in a red alga Porphyra yezoensis by particle bombardment, a stable transformation system has yet to establish in any marine red macrophytes. One of the reasons of the difficulty in genetic transformation in red algae is the lack of systems to select and isolate transformed cells from gametophytic blades. Thus, toward the establishment of the stable transformation system in P. yezoensis, we have developed a procedure by which transiently transformed gametophytic cells were prepared from particle bombarded-gametophytic blade as regeneratable protoplasts. Using mixture of marine bacterial enzymes, yield of protoplasts was high as reported elsewhere; however, these protoplasts did not develop. In contrast, protoplasts prepared from gametophytes treated with allantoin were normally developed, in which the overexpression of a â-glucuronidase reporter gene had no effect on the regeneration of protoplasts. Therefore, the use of allantoin in protoplast preparation sheds a new light on the realization of an efficient isolation and selection of study transformed cells from gametophytic blades.

Isolation and fusion of protoplasts from the phytopathogenic fungus Sclerotium rolfsii (Sacc. )

Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x10(5) /g mycelia and 2.8x10(5) /g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30 percent (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/microg of DNA in 1 x 107 protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

Acquiring homozygous tetraploid germplasm by PEG-mediated protoplast fusion of Rhodiola sachalinensis / 中国中药杂志

<p><b>OBJECTIVE</b>To acquire homozygous tetraploid germplasm of Rhodiola sachalinensis.</p><p><b>METHOD</b>PEG-mediated protoplast fusions were conducted using callus of Rh. sachalinensis as materials. Protoplast fusion products were embedded and cultured in low-density, low-melting-point agar and marked according to the protoplast size, and single-celled sister lines were established to acquire genetically homozygous tetraploid germplasm.</p><p><b>RESULT</b>R(D) and R(M) of newborn daughter cells or protoplasm, metaphase cells or protoplasm were approximately in line with the formula R(D) = 0.793 7R(M). The change range in diameter of the diploid cells without fusion, two protoplasts fusion product were: 16.7 microm < or = R < 21.3 microm, 21.0 microm < or = R' < 26.8 microm respectively. There is an overlap between the two diameter ranges. The protoplast inoculation density of 1 x 10(4) cells x mL(-1) was appropriate when protoplasts were anchored by low-intensity, low-melting-point agar. Under the conditions of this density, plating efficiency was high and single cell origin of the sister lines microclones grew rapidly, and it was easy to mark the single cell microclones, and separate from each other to subculture. The chromosome counts results showed that chromosome numbers of diploid and tetraploid of single cell lines were 26 and 52, respectively. The result from flow cytometry assay showed that there is no presence of chimerism in single-cell regeneration plantlets.</p><p><b>CONCLUSION</b>The results of this study provide a scientific basis for polyploid breeding of Rh. sachalinensis.</p>

Comparisons of different methods for virus-elimination of edible fungi / 病毒学报

Four dsRNA bands were extracted from Pleurotus ostreatus TD300 by the dsRNA isolation technique with sizes of 8.2 kb, 2.5 kb, 2.1 kb, and 1.1 kb, respectively. Four virus-eliminated methods, i. e. hyphal tips cut (HTC), protoplast regeneration (PR), single spore hybridization (SSH), and frozen and lyophilized (FL), were applied to prepare virus-eliminated strains, and one virus-eliminated strain was selected for each virus-elimination method. The virus-eliminated strains were named as HTC8, PR15, FL01, and SSH11, respectively. There were low concentration of 8.2 kb dsRNA remained in HTC8, as well as low concentration of 8.2 kb and 2.5 kb dsRNA remained in FL01. However, no dsRNA remained in PR15 and SSH11. The hyphal growth rate and laccase activity of the virus-eliminated strains increased, especially HTC8 and PR15, whose hyphal growth rate was higher by 22.73% and 18.18%, and laccase activities higher by 145.83% and 134.38% than that of the original strain, respectively. The conclusion is that hyphal tips cut and protoplast regeneration are suitable to prepare virus-eliminated strains of edible fungi.

Glass bead transformation method for gram-positive bacteria

A simple, inexpensive and reproducible transformation method was developed for Gram-positive bacteria. It was based on agitation of bacterial protoplasts with glass beads in the presence of DNA and polyethylene glycol. By using this method, introduction of pGK12 into protoplasts of several strains of Gram-positive bacteria was achieved.

Protoplast mutagenesis for improving beta-glucosidase production of Aspergillus niger / 生物工程学报

The aims of this research were to isolate a Aspergillus niger strain with higher beta-glucosidase activity. We utilized the beta-glucosidase producing strain Aspergillus niger CGMCC 3.316 as the original strain to first obtain a mutant 3-3M through ultraviolet irradiation. Then we studied the conditions of protoplast release and regeneration for strain 3-3M. We treated the protoplasts of strain 3-3M via ultraviolet irradiation and obtained another isolated mutant 60B-3D. The strain 60B-3D showed much higher beta-glucosidase production than the original strain and 3-3M strain. The beta-glucosidase activity of strain 60B-3D was 23.4 IU/mL, with an improvement of 39% compared with the original strain, and 23% compared with strain 3-3M. We also studied the fermentation process of strain 60B-3D, and compared it with the original strain and strain 3-3M. We found the strain 60B-3D exhibited an improvement in xylanase production. The comparison results also showed that the strain 60B-3D secreted more protein. These results were beneficial for producing beta-glucosidase through this productive mutant.