The usage of snapdragon Delila (Del) gene as a visible selection marker for the antibiotic-free transformation system

2015 ◽  
Vol 58 (2) ◽  
pp. 110-116 ◽  
Author(s):  
Aung Htay Naing ◽  
Ki Byung Lim ◽  
Chang Kil Kim
Keyword(s):  
Selection Marker ◽  
Transformation System

scholarly journals Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

2012 ◽  
Vol 78 (22) ◽  
pp. 7968-7976 ◽  
Author(s):  
Jun-Wei Xu ◽  
Yi-Ning Xu ◽  
Jian-Jiang Zhong
Keyword(s):  
Ganoderma Lucidum ◽  
Coenzyme A ◽  
Catalytic Domain ◽  
Selection Marker ◽  
Transformation System ◽  
Protein Subunit ◽  
Ganoderic Acid ◽  
Content Type ◽  
Ganoderic Acids ◽  
Coenzyme A Reductase

ABSTRACTGanoderic acids produced byGanoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification ofG. lucidumis difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system forG. lucidumwas developed for the first time using mutatedsdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncatedG. lucidumgene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using theAgrobacterium tumefaciens-mediated transformation system. The results showed that the mutatedsdhBsuccessfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomyceteG. lucidumis a promising system to achieve metabolic engineering of the ganoderic acid pathway.

Development of an integrative transformation system for the opportunistic pathogenic yeastCandida lusitaniae usingURA3 as a selection marker

Yeast ◽  
2004 ◽  
Vol 21 (2) ◽  
pp. 95-106 ◽  
Author(s):  
Fabienne François ◽  
Florence Chapeland-Leclerc ◽  
Jean Villard ◽  
Thierry Noël
Keyword(s):  
Selection Marker ◽  
Transformation System ◽  
Integrative Transformation ◽  
Opportunistic Pathogenic

scholarly journals Integrative Transformation System for the Metabolic Engineering of the Sphingoid Base-Producing Yeast Pichia ciferrii

2003 ◽  
Vol 69 (2) ◽  
pp. 812-819 ◽  
Author(s):  
Jung-Hoon Bae ◽  
Jung-Hoon Sohn ◽  
Chang-Seo Park ◽  
Joon-Shick Rhee ◽  
Eui-Sung Choi
Keyword(s):  
Metabolic Engineering ◽  
De Novo ◽  
Integration Site ◽  
Fold Increase ◽  
Selection Marker ◽  
Serine Palmitoyltransferase ◽  
Transformation System ◽  
Wild Type ◽  
Gene Encoding ◽  
Integrative Transformation

ABSTRACT We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P. ciferrii as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/μg of DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii and overexpressed under the control of the P. ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.

scholarly journals Generation of Transgenic Rice without Antibiotic Selection Marker through Agrobacterium-mediated Co-transformation System

2012 ◽  
Vol 22 (9) ◽  
pp. 1152-1158
Author(s):  
Soo-Kwon Park ◽  
Tack-Min Kwon ◽  
Jong-Hee Lee ◽  
Dong-Jin Shin ◽  
Woon-Ha Hwang ◽  
...  
Keyword(s):  
Transgenic Rice ◽  
Selection Marker ◽  
Transformation System ◽  
Antibiotic Selection

Development of a nuclear transformation system with a codon-optimized selection marker and reporter genes in Pyropia yezoensis (Rhodophyta)

2014 ◽  
Vol 26 (4) ◽  
pp. 1863-1868 ◽  
Author(s):  
Ryo Hirata ◽  
Toshiki Uji ◽  
Satoru Fukuda ◽  
Hiroyuki Mizuta ◽  
Asao Fujiyama ◽  
...  
Keyword(s):  
Reporter Genes ◽  
Pyropia Yezoensis ◽  
Selection Marker ◽  
Nuclear Transformation ◽  
Transformation System

scholarly journals A simple and rapid protocol for the genetic transformation of Ensete ventricosum

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Jonathan Matheka ◽  
Jaindra Nath Tripathi ◽  
Ibsa Merga ◽  
Endale Gebre ◽  
Leena Tripathi
Keyword(s):  
Disease Resistance ◽  
Plant Regeneration ◽  
Genetic Transformation ◽  
Marker Gene ◽  
Selection Marker ◽  
Tropical Species ◽  
Transformation System ◽  
Regeneration System ◽  
Ensete Ventricosum ◽  
Multiple Buds

Abstract Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.

scholarly journals Transformation system for prototrophic industrial yeasts using theAUR1gene as a dominant selection marker

FEBS Letters ◽  
1998 ◽  
Vol 425 (1) ◽  
pp. 117-122 ◽  
Author(s):  
Takashi Hashida-Okado ◽  
Atsuko Ogawa ◽  
Ikunoshin Kato ◽  
Kazutoh Takesako
Keyword(s):  
Selection Marker ◽  
Transformation System ◽  
Industrial Yeasts

scholarly journals Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1293
Author(s):  
Lakkakula Satish ◽  
Madhu Kamle ◽  
Guy Keren ◽  
Chandrashekhar D. Patil ◽  
Galit Yehezkel ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Optical Density ◽  
Fluorescent Protein ◽  
Basic Knowledge ◽  
Selection Marker ◽  
Transformation System ◽  
Intensive Farming ◽  
Terfezia Boudieri ◽  
Desert Truffles

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 μM of acetosyringone, attaining a bacterial optical density of 0.3 OD600; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.

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