scholarly journals Agrobacterium-mediated transformation of potato with human lactoferrin gene

2019 ◽  
Vol 25 ◽  
pp. 184-189
Author(s):  
A. Yu. Buziashvili ◽  
A. I. Yemets
Keyword(s):  
Fungal Pathogens ◽  
Transgenic Potato ◽  
Transformation Frequency ◽  
Potato Cultivars ◽  
Human Lactoferrin ◽  
Chain Reaction ◽  
Agrobacterium Mediated Transformation ◽  
Lactoferrin Gene ◽  
Polymerase Chain ◽  
Resistance To Phytopathogens

Aim. Obtaining of the new Ukrainian potato cultivars with complex resistance to phytopathogens. Methods. Agrobacterium-mediated transformation of potato cultivars Vernisage and Zarevo with human lactoferrin gene possessing antimicrobial activity was carried out. For transformation, supervirulent A. tumefaciens strain EHA 105 with hLf gene and gene for the resistance to kanamycine was used. Selection was carried out for 3 months on MSK-S1 and MSK-S2 media supplemented with 100 mg/l kanamycine. Integration of lactoferrin gene in transgenic plant lines was confirmed by polymerase chain reaction with specific for hLf gene primers. Results. After selection, potato lines with the resistance to kanamycine were obtained. The transgenic potato lines with integrated into their genomes hLf gene were obtained. Integration of lactoferrin gene was confirmed for 1 line of Zarevo cultivar and 3 lines of Vernisage cultivar. Transformation frequency was of 6.8% for cv. Vernisage and 6.25% for cv. Zarevo. Conclusions. Selected potato lines would be used in further studies on their resistance to bacterial and fungal pathogens. Keywords: human lactoferrin gene hLf, Solanum tuberosum, Agrobacterium-mediated transformation, transgenic plants.

DNA Isolation from Recalcitrant Materials Such as Tree Roots, Bark, and Forest Soil for the Detection of Fungal Pathogens by Polymerase Chain Reaction

1998 ◽  
Vol 262 (1) ◽  
pp. 79-82 ◽  
Author(s):  
Günther Bahnweg ◽  
Steffen Schulze ◽  
Evelyn M. Möller ◽  
Hilkea Rosenbrock ◽  
Christian Langebartels ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Forest Soil ◽  
Fungal Pathogens ◽  
Dna Isolation ◽  
Chain Reaction ◽  
Tree Roots ◽  
Polymerase Chain

scholarly journals Polymerase Chain Reaction Detection and Phylogenetic Characterization of an Agent Associated with Yellow Vine Disease of Cucurbits

1998 ◽  
Vol 88 (5) ◽  
pp. 428-436 ◽  
Author(s):  
Francisco J. Avila ◽  
Benny D. Bruton ◽  
Jacqueline Fletcher ◽  
J. L. Sherwood ◽  
Sam D. Pair ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleotide Sequence ◽  
16S Rdna ◽  
Fungal Pathogens ◽  
Pcr Amplification ◽  
Diagnostic Techniques ◽  
Chain Reaction ◽  
Phylogenetic Characterization ◽  
South Central ◽  
Polymerase Chain

Diagnosis of yellow vine disease (YVD) in cucurbits, an important disease in the south-central United States, relies on external symptom appearance, phloem discoloration, and the presence of bacterium-like organisms (BLOs) in phloem. Polymerase chain reaction (PCR) amplification of BLO nucleotide sequences was explored as a means to improve diagnostic techniques. PCR, using a primer pair based on sequences of the citrus-greening BLO, amplified a 0.15-kilobase (kb) fragment from the DNA of symptomatic plants, but not from that of asymptomatic plants. Its nucleotide sequence suggested that the DNA amplified was of pro-karyotic origin. A primer pair, designed to amplify nonspecific prokaryotic 16S rDNA, amplified a 1.5-kb DNA fragment in both the symptomatic and asymptomatic plants. The 1.5-kb fragment from the asymptomatic plants corresponded to chloroplast 16S rDNA, and the band from the symptomatic plants was composed of 16S rDNAs from both chloroplasts and a prokaryote. The nucleotide sequence of the prokaryotic DNA was determined and used to design three primers (YV1, YV2, and YV3). Fragments of 0.64 and 1.43 kb were amplified with primers YV1-YV2 and primers YV1-YV3, respectively, from symptomatic plants. Neither primer set yielded fragments from asymptomatic plants, unrelated bacteria, or selected soilborne fungal pathogens of cucurbits. Phylogenetic analysis indicated that the prokaryote is a gamma-3 proteobacterium. The consistent association of the 0.64- and 1.43-kb fragments with symptomatic plants suggests that the gamma-3 proteobacterium may be the causal agent of YVD of cantaloupe, squash, and watermelon.

scholarly journals Detection of Gaeumannomyces graminis Varieties Using Polymerase Chain Reaction with Variety-Specific Primers

Plant Disease ◽  
2000 ◽  
Vol 84 (9) ◽  
pp. 947-951 ◽  
Author(s):  
H. M. Fouly ◽  
H. T. Wilkinson
Keyword(s):  
Polymerase Chain Reaction ◽  
Fungal Pathogens ◽  
Gaeumannomyces Graminis ◽  
Universal Primers ◽  
Middle Region ◽  
Chain Reaction ◽  
Specific Primers ◽  
Grass Roots ◽  
Polymerase Chain ◽  
Take All

The polymerase chain reaction (PCR) was used for detection of Gaeumannomyces graminis, the causal agent of take-all disease in wheat, oats, and turfgrass. NS5 and NS6 universal primers amplified the middle region of 18S ribosomal DNA of Gaeumannomyces species and varieties. Primers GGT-RP (5′ TGCAATGGCTTCGTGAA 3′) and GGA-RP (5′ TTTGTGTGTGAC CATAC 3′) were developed by sequence analysis of cloned NS5-NS6 fragments. The primer pair NS5:GGT-RP amplified a single 410-bp fragment from isolates of G. graminis var. tritici, a single 300-bp fragment from isolates of G. graminis var. avenae, and no amplification products from isolates of G. graminis var. graminis or other species of Gaeumannomyces. The primer pair NS5:GGA-RP amplified a single 400-bp fragment from isolates of varieties tritici and avenae. Two sets of primer pairs (NS5:GGT-RP and NS5:GGA-RP) were used in PCR reactions to detect and identify the varieties tritici and avenae either colonizing wheat, oats, or grass roots, or in culture. No amplification products were observed using DNA extracted from plants infected with eight other soilborne fungal pathogens or from uninoculated plants.

scholarly journals Overexpression of Soybean-Derived Lunasin in Wheat and Assessment of Its Anti-Proliferative Activity in Colorectal Cancer HT-29 Cells

2020 ◽  
Vol 21 (24) ◽  
pp. 9594
Author(s):  
Xin Fan ◽  
Peiyou Qin ◽  
Yuqiong Hao ◽  
Huimin Guo ◽  
Christophe Blecker ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Proliferative Activity ◽  
Transgenic Wheat ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Chain Reaction ◽  
Apoptosis Pathway ◽  
Agrobacterium Mediated Transformation ◽  
Polymerase Chain ◽  
Ht 29

Lunasin is a soybean-derived peptide that exhibits anticancer bioactivity in different cancer cells and has been identified in different plants. However, recent studies revealed through molecular and chemical analyses that lunasin was absent in wheat and other cereals. In this study, the soybean-derived lunasin was cloned into pCAMBIA3300 and we transferred the expression vector into wheat via an Agrobacterium-mediated transformation. The identification of transgenic wheat was detected by polymerase chain reaction, Western blot analysis, and ultra-performance liquid chromatography with tandem mass spectrometry. An enzyme-linked immunosorbent assay showed that lunasin content in transgenic wheat L32-3, L32-6, and L33-1 was 308.63, 436.78, and 349.07 µg/g, respectively, while lunasin was not detected in wild-type wheat. Lunasin enrichment from transgenic wheat displayed an increased anti-proliferative activity compared with peptide enrichment from wild-type wheat in HT-29 cells. Moreover, the results of a real-time quantitative polymerase chain reaction showed a significant elevation in p21, Bax, and caspase-3 expression, while Bcl-2 was significantly downregulated. In conclusion, soybean-derived lunasin was successfully expressed in wheat via Agrobacterium-mediated transformation and may exert anti-proliferative activity by regulating the apoptosis pathway in HT-29 cells, which provides an effective approach to compensate for the absence of lunasin in wheat.

scholarly journals Quantitative Multiplexed Detection of Common Pulmonary Fungal Pathogens by Labeled Primer Polymerase Chain Reaction

2014 ◽  
Vol 138 (11) ◽  
pp. 1474-1480 ◽  
Author(s):  
Zhengming Gu ◽  
Daelynn R. Buelow ◽  
Ruta Petraitiene ◽  
Vidmantas Petraitis ◽  
Thomas J. Walsh ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Fungal Pathogens ◽  
Bronchoalveolar Lavage Fluid ◽  
Fungal Infections ◽  
Lavage Fluid ◽  
Quantitative Detection ◽  
Target Sequence ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Primer Polymerase Chain Reaction

Context Invasive fungal infections are an important cause of morbidity and mortality among immunocompromised patients. Objective To design and evaluate a multiplexed assay aimed at quantitative detection and differentiation of the 5 molds that are most commonly responsible for pulmonary infections. Design Using labeled primer polymerase chain reaction chemistry, an assay was designed to target the 5.8S and 28S ribosomal RNA genes of Aspergillus spp, Fusarium spp, Scedosporium spp, and members of the order Mucorales (Rhizopus oryzae, Rhizopus microsporus, Cunninghamella bertholletiae, Mucor circinelloides, Lichtheimia corymbifera, and Rhizomucor pusillus). This assay was split into 2 multiplexed reactions and was evaluated using both samples seeded with purified nucleic acid from 42 well-characterized clinical fungal isolates and 105 archived samples (47 blood [45%], 42 bronchoalveolar lavage fluid [40%], and 16 tissue [15%]) collected from rabbit models of invasive pulmonary fungal infections. Results Assay detection sensitivity was less than 25 copies of the target sequence per reaction for Aspergillus spp, 5 copies for Fusarium spp and Scedosporium spp, and 10 copies for the Mucorales. The assay showed quantitative linearity from 5 × 101 to 5 × 105 copies of target sequence per reaction. Sensitivities and specificities for bronchoalveolar lavage fluid, tissue, and blood samples were 0.86 and 0.99, 0.60 and 1.00, and 0.46 and 1.00, respectively. Conclusions Labeled primer polymerase chain reaction permits rapid, quantitative detection and differentiation of common agents of invasive fungal infection. The assay described herein shows promise for clinical implementation that may have a significant effect on the rapid diagnosis and treatment of patients' severe infections caused by these pulmonary fungal pathogens.

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