Selectable marker-free transgenic barley producing a high level of cellulase (1,4-?-glucanase) in developing grains

ABSTRACTWe report here a novel selectable marker for the hyperthermophilic crenarchaeonSulfolobus islandicus. The marker cassette is composed of thesac7dpromoter and thehmggene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Psac7d-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting thepyrEFgene of the expression vector pSeSD for Psac7d-hmgwith which theSulfolobusexpression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization ofSulfolobustransformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His6-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEFandhmg) was subsequently exploited for genetic analysis. AherAmerodiploid strain ofS. islandicuswas constructed usingpyrEFmarker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from theherAmerodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability ofS. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.

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A Site-Specific Recombinase-Based Method to Produce Antibiotic Selectable Marker Free Transgenic Cattle

PLoS ONE ◽

10.1371/journal.pone.0062457 ◽

2013 ◽

Vol 8 (5) ◽

pp. e62457 ◽

Cited By ~ 22

Author(s):

Yuan Yu ◽

Yongsheng Wang ◽

Qi Tong ◽

Xu Liu ◽

Feng Su ◽

...

Keyword(s):

Selectable Marker ◽

Site Specific ◽

Transgenic Cattle ◽

Marker Free ◽

A Site

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Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all regenerants

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-010-9737-1 ◽

2010 ◽

Vol 102 (3) ◽

pp. 329-336 ◽

Cited By ~ 25

Author(s):

A. Ballester ◽

M. Cervera ◽

L. Peña

Keyword(s):

Selectable Marker ◽

Pcr Analysis ◽

Marker Free

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Inheritance of NmDef02 synthetic defensin gene and hpt selectable marker in four successive generations of transgenic rice (Oryza sativa, cv. J-104)

International Journal of Current Research in Biosciences and Plant Biology ◽

10.20546/ijcrbp.2021.809.005 ◽

2021 ◽

Vol 8 (9) ◽

pp. 34-41

Author(s):

Maylin Pérez-Bernal ◽

Magali Delgado ◽

Daymí Abreu ◽

Onel Valdivia ◽

Raúl Armas

Keyword(s):

Transgenic Rice ◽

Selectable Marker ◽

Marker Gene ◽

Rice Cultivar ◽

Future Research ◽

Maturation Stage ◽

Defensin Gene ◽

Regenerated Plants ◽

Marker Free ◽

Successive Generations

The evaluation of the inheritance and stability of the transgenes is essential for the application of transgenic plants in agriculture. In this work, we studied the inheritance of two transgenes in T1, T2, T3 and T4 rice progenies. Transgenic rice plants (cv. J-104) was obtained by biolistic method using a synthetic defensin gene (NmDef02) and hpt as selectable marker gene, co-transformed in a 4:1 proportion, respectively. Regenerated plants were acclimated under natural conditions. The study started from the primary transformants that fulfilled the agronomic characters reported by the experts for the J-104 rice cultivar in the maturation stage. The integration and relative expression of NmDef02 in T1 plants was verified by Southern blot and qRT-PCR, respectively. The inheritance of transgenes over four generations was analyzed by PCR. The following transgene combinations were identified: NmDef02(+)hpt(+), NmDef02(+)hpt(-) and NmDef02(-)hpt(-). The most advantageous combination was NmDef02(+)hpt(-), which corresponded to the marker-free plants harboring the gene of interest. The inheritance of NmDef02 was confirmed in T1 and T2 progenies, but in some T3 and T4 lines the loss of this gene was verified. This inheritance analysis provided information concerning the transgenic population, but the mechanisms that destabilize the inheritance of the gene of interest will be the goal of future research.

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