Construction of expression vectors of the melon resistance gene Fom-2 and genetic transformation

2018 ◽  
Vol 51 (1) ◽  
Author(s):  
Xiaomei Li ◽  
Liu Liu ◽  
Xiangyu Liu ◽  
Yan Hou ◽  
Bingyin Xu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Resistance Gene ◽  
Expression Vectors

Genetic transformation of Nannochloropsis oculata with a bacterial phleomycin resistance gene as dominant selective marker

2016 ◽  
Vol 15 (2) ◽  
pp. 351-356 ◽  
Author(s):  
Xiaolei Ma ◽  
Kehou Pan ◽  
Lin Zhang ◽  
Baohua Zhu ◽  
Guanpin Yang ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Resistance Gene ◽  
Nannochloropsis Oculata ◽  
Selective Marker

scholarly journals Genetic Transformation of the Obligate Parasite Plasmodiophora brassicae

2013 ◽  
Vol 103 (10) ◽  
pp. 1052-1057 ◽  
Author(s):  
J. Feng ◽  
Sheau-Fang Hwang ◽  
S. E. Strelkov
Keyword(s):  
Genetic Transformation ◽  
Fluorescent Protein ◽  
Plasmodiophora Brassicae ◽  
Expression Vectors ◽  
Obligate Parasite ◽  
Putative Transformants ◽  
Resting Spores ◽  
Selection Step ◽  
Protoplast Preparation ◽  
Green Fluorescent

A protocol for genetic transformation of the obligate parasite Plasmodiophora brassicae, causal agent of clubroot of crucifers, was developed. In this protocol, protoplast preparation was superseded with lithium acetate treatment and the selection step was omitted. In two independent experiments, germinating resting spores of P. brassicae were transformed by two fungal expression vectors containing either a green fluorescent protein (gfp) gene or a hygromycin resistance (hph) gene. Putative transformants were produced from both transformations, with ≈50% of the obtained galls containing resting spores from which transforming DNA could be detected by polymerase chain reaction (PCR). PCR, quantitative PCR (qPCR), and genome walking conducted on selected transformants indicated that the transforming DNA was intergraded into the P. brassicae genome. Transcript of hph but not gfp was detected by reverse-transcription qPCR from selected transformants. From all galls produced by transformants, no GFP activity could be identified. Verified transformants were inoculated on canola and new galls were generated. PCR and qPCR analyses based on these galls indicated that transforming DNA was still resident in P. brassicae. This is the first report on genetic transformation of P. brassicae. The information and data generated from this study will facilitate research in multiple areas of the clubroot pathosystem.

scholarly journals Alpha-tubulin promoter from Chlorella vulgaris allows genetic transformation of green coccoid microalga

2020 ◽  
Author(s):  
Raquel Fernández-Rodríguez ◽  
Giovanni Garro-Monge ◽  
Maritza Guerrero-Barrantes ◽  
Olman Gómez-Espinoza
Keyword(s):  
Genetic Transformation ◽  
Resistance Gene ◽  
Chlorella Vulgaris ◽  
Heterologous Gene Expression ◽  
Antibiotic Resistance Gene ◽  
Gc Content ◽  
Proximal Promoter ◽  
Start Codon ◽  
Upstream Region ◽  
Alpha Tubulin

Microalgae have become a feasible platform for high-value recombinant protein production. Although diverse sets of genetic tools for microalgae transformation have been developed, some critical elements, such as endogenous promoters, need to be improved for increasing transgene expression levels after genetic transformation. This work aims to evaluate a sequence from the 5′ upstream region of the alpha-tubulin gene from Chlorella vulgarisas a promoter for the expression of an antibiotic resistant gene in Chlorella sorokiniana. Using in silicoanalysis it was possible to identify a proximal promoter corresponding to 281 nucleotides upstream of the ATG start codon, which possessed 9 potential cis-regulatory element, including TATA Box and CAAT Box. The proximal promoter sequencewas used to drive the expression of a streptomycin-resistance gene (aada), previous optimization of its codon usage. The codon optimization of the aadasequence allowed it to obtain a GC content of 69.8% (compared to 53% of the original sequence), which increases its similarity with Chlorellasp. genomes (67.2%). Both genetic elements were cloned into the pUC57-Kan vector and transformed into C. sorokiniana cells by electroporation. The microalgae transgenic colonies were identified through culture in selective medium and PCR. Our results proved the capacity of the Chlorella vulgaris alpha-tubulin promoter to express a foreign antibiotic-resistance gene in C. sorokinianacells. Research of endogenous promoting sequences is essential in order to accomplish an efficient heterologous gene expression, especially in microalgae used for industrial production like those from Chlorella genus.  

Stable genetic transformation of Arabidopsis thaliana by Agrobacterium inoculation in planta

1994 ◽  
Vol 5 (4) ◽  
pp. 551-558 ◽  
Author(s):  
Seok So Chang ◽  
Soon Ki Park ◽  
Byung Chul Kim ◽  
Bong Joong Kang ◽  
Dal Ung Kim ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Genetic Transformation ◽  
In Planta
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