Polycistronic strategy for cyanobacterial expression vector construction: Co-transcription of a human gene and a selective marker gene

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d / r) or inverted-repeat (i / r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i / r T-DNA insertions is a primary inducer of transgene silencing in plants.

Download Full-text

Cloning of strawberry FaEtr2 gene and its plant expression vector construction for antisense RNA

Frontiers of Agriculture in China ◽

10.1007/s11703-009-0017-y ◽

2009 ◽

Vol 3 (1) ◽

pp. 55-59 ◽

Cited By ~ 1

Author(s):

Chunli Song ◽

Junlian Ma ◽

Xia Tang ◽

Zide Zhang ◽

Pingping Zhou ◽

...

Keyword(s):

Antisense Rna ◽

Expression Vector ◽

Plant Expression ◽

Plant Expression Vector ◽

Vector Construction

Download Full-text

Molecular cloning and expression vector construction of bovine TRIM28

Genetics and Molecular Research ◽

10.4238/gmr.15028793 ◽

2016 ◽

Vol 15 (2) ◽

Author(s):

X. Ma ◽

Z.C. Zhai ◽

M.L. Zhang ◽

B.H. Song ◽

Y.R. Zhu ◽

...

Keyword(s):

Molecular Cloning ◽

Expression Vector ◽

Cloning And Expression ◽

Vector Construction

Download Full-text

Characterization of plasmid pASV479 from Bifidobacterium pseudolongum subsp. globosum and its use for expression vector construction

Plasmid ◽

10.1016/j.plasmid.2007.02.004 ◽

2007 ◽

Vol 58 (2) ◽

pp. 140-147 ◽

Cited By ~ 18

Author(s):

A. Sangrador-Vegas ◽

C. Stanton ◽

D. van Sinderen ◽

G.F. Fitzgerald ◽

R.P. Ross

Keyword(s):

Expression Vector ◽

Vector Construction

Download Full-text

Cloning of a flower-specific expression promoter from Arabidopsis thaliana and its plant expression vector construction

Forestry Studies in China ◽

10.1007/s11632-010-0408-4 ◽

2010 ◽

Vol 12 (4) ◽

pp. 201-205

Author(s):

Bing-you Fan ◽

Shui-ping Gao ◽

Xiao-gai Hou ◽

Guo-an Shi

Keyword(s):

Arabidopsis Thaliana ◽

Expression Vector ◽

Specific Expression ◽

Plant Expression ◽

Plant Expression Vector ◽

Vector Construction

Download Full-text

BINARY VECTOR CONSTRUCTION OF KAPPA(κ)-CARRAGEENASE GENE AND TRANSFORMATION TO Agrobacterium tumefaciens AS MEDIATOR FOR SEAWEED TRANSGENIC GENERATION

Jurnal Ilmu dan Teknologi Kelautan Tropis ◽

10.28930/jitkt.v8i1.13602 ◽

2016 ◽

Vol 8 (1) ◽

Author(s):

Muh Alias L. Rajamuddin ◽

. Alimuddin ◽

Utut Widyastuti ◽

Enang Harris ◽

Emma Suryati

Keyword(s):

Agrobacterium Tumefaciens ◽

Heat Shock ◽

Expression Vector ◽

Gene Sequence ◽

Binary Vector ◽

Pcr Analysis ◽

Vector Construction ◽

Dna Fragment ◽

Dna Template ◽

Parental Mating

Increasing of kappa (κ)-carrageenan content in Kappaphycus alvarezii seaweed is potentially be achieved by applying transgenesis technology. This study was performed to obtain a construction of κ-Carrageenase gene and Agrobacterium tumefaciens to carry those construction genes. The κ-Carrageenase (κ-Car) gene was involved in κ-carrageenan biosynthesis. The κ-Car gene sequence was ligated between the 35S CaMV promoter and tNos terminator sequences to generate pMSH/κ-Car expression vector. Transformation of pMSH/κ-Car plasmid to Escherichia coli was performed by heat-shock method, and to Agrobacterium tumefaciens by tri-parental mating method. The results showed that several colonies of E. coli and A. tumefaciens grew in the selective culture mediums containing antibiotic. PCR analysis using primers 35S-Forward and tNos-Reverse with DNA template from those bacterial colonies resulted DNA fragment of about 2,000 bp, the same as the total length of 35S CaMV promoter, κ-Car gene and tNos terminator sequences. Therefore, the construction of pMSH/κ-Car gene was succeeded and a colony of A. tumefaciens transformant carrying pMSH/κ-Car plasmid was successfully produced. Keywords: Agrobacterium tumefaciens, kappa(κ)-Carrageenase gene, transgenesis, vector

Download Full-text

Cloning of XET gene from Anthocephalus chinensis and its plant expression vector construction

Forestry Studies in China ◽

10.1007/s11632-010-0015-4 ◽

2010 ◽

Vol 12 (2) ◽

pp. 79-84 ◽

Cited By ~ 1

Author(s):

Sheng-jun Ma ◽

Song-lin Zhu ◽

Wei Li ◽

Kun-xi Ouyang ◽

Na Li ◽

...

Keyword(s):

Expression Vector ◽

Plant Expression ◽

Plant Expression Vector ◽

Vector Construction

Download Full-text

Tightly regulated and high level expression vector construction for Escherichia coli BL21 (DE3)

Journal of Industrial and Engineering Chemistry ◽

10.1016/j.jiec.2015.07.011 ◽

2015 ◽

Vol 31 ◽

pp. 367-373 ◽

Cited By ~ 2

Author(s):

Amit Kumar Chaudhary ◽

Eun Yeol Lee

Keyword(s):

Escherichia Coli ◽

Expression Vector ◽

High Level Expression ◽

Escherichia Coli Bl21 ◽

Vector Construction ◽

High Level

Download Full-text

Combination of site-specific recombination and a conditional selective marker gene allows for the production of marker-free tobacco plants

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-013-0396-x ◽

2013 ◽

Vol 116 (2) ◽

pp. 205-215 ◽

Cited By ~ 11

Author(s):

R. C. García-Almodóvar ◽

C. Petri ◽

I. M. G. Padilla ◽

L. Burgos

Keyword(s):

Marker Gene ◽

Selective Marker ◽

Tobacco Plants ◽

Site Specific ◽

Site Specific Recombination ◽

Marker Free

Download Full-text

ISOLATION OF Β-ACTIN PROMOTER DERIVED FROM WALKING CATFISH (CLARIAS BATRACHUS)

KnE Life Sciences ◽

10.18502/kls.v1i0.105 ◽

2015 ◽

Vol 2 (1) ◽

pp. 201

Author(s):

Ibnu Dwi Buwono ◽

Nono Carsono ◽

Yuniar Mulyani ◽

Mochamad Untung Kurnia Agung

Keyword(s):

Growth Hormone ◽

Expression Vector ◽

Fish Growth ◽

Clarias Batrachus ◽

Growth Hormone Gene ◽

Economic Traits ◽

Food Material ◽

Vector Construction ◽

Reverse Primer ◽

Actin Promoter

Fish growth improvement as economic traits can be solved through fish transgenic production. Growth hormone gene is inserted into transgenic vector construction to over-express fish growth. The promoter as a part of the expression vector has an important role in its regulation. The use of promoter which is derived from mammalian or virus (such as CMV/ cytomegalovirus) in the expression vector, in specific goal as food material, has customer resistant rather than a promoter which is derived from in- sibling species. Beside of it, transgene expression level when using in-sibling promoter showed higher than using mammalian or viral promoter. The β-actin promoter is screened from walking catfish pituitary genome DNA using primers: pBA-cy-F (5’- GTGWGTGACGCYGGACCAAATC-3’) as forward primer and pBA-cy-R (5’- CCATRTCRTCCCAGTTGGTSACAAT-3’) as reverse primer, produced an amplicon of 1,7 kb in length. Sequence analysis using TF BindTM indicated transcription factor elements: TATA box, CCAAT box, enhancer (CAAT), and CarGG (CAAATGG) motif. This result showed that promoter which is obtained from this research is useful in construction of all catfish growth hormone vector expression catfish transgenic production. Keywords: β-actin promoter, growth hormone, expression vector, walking catfish (Clarias batrachus)

Download Full-text