Expression of modified 7SL RNA gene in transgenic Solanum tuberosum plants

Abstract

A modified plant 7SL RNA gene from Arabidopsis thaliana designated AHIIA63M was introduced into potato plants via Agrobacterium-mediated transformation. No transgenic plants could be obtained using pGPTV-based binary vectors where AHIIA63M gene driven by polIII promoter was located close to the polII promoter of the selection gene. Special binary vectors with matrix attachment region (MAR) elements had to be used for transformation to insulate polII and polIII promoters within T-DNA. The level of AHIIA63M RNA in transgenic plants was lower than the levels of transcripts of transgenes driven by RNA polymerase II. The level of AHIIA63M transcript in transgenic potato plants was tissue specific. The highest expression was detected in roots and gynoecium and the lowest in tubers. Moreover, non-specific promoter activity within the MAR element was revealed. This activity contributed to AHIIA63M transcription. This is the first report of expression of a modified 7SL RNA gene in transgenic plants and promoter activity within the MAR element.Additional key words: RNA polymerase III, polIII expression cassette, MAR element.

Combinatorial analysis of lupulin gland transcription factors from R2R3Myb, bHLH and WDR families indicates a complex regulation of chs_H1 genes essential for prenylflavonoid biosynthesis in hop (Humulus LupulusL.)

“…Gel-purified fragments were then fused to the 35S CaMV promoter by cloning into the corresponding unique sites of the intermediary vector pLV68 [ 16 ]. The whole TF cassettes containing the 35S CaMV promoter were excised using restrictases Asc I and Pac I and cloned in the plant vector pLV07 as described by Vrba and Matoušek [ 73 ]. These vectors were introduced in the A. tumefaciens strain LBA 4404 by the freeze-thaw method [ 74 ] and used for leaf infiltration of N. benthamiana or P. hybrida leaves as described previously [ 16 ].…”

Sequence Analysis of a “True” Chalcone Synthase (chs_H1) Oligofamily from hop (Humulus lupulus L.) and PAP1 Activation of chs_H1 in Heterologous Systems

“…A NcoI-BamHI PAP1 fragment containing the complete coding region was cloned in the frame between the CaMV 35S promoter and the polyA signal of the vector pRT-100 (21). This PAP1 expression cassette was then excised with HindIII and cloned into the HindIII site of the binary vector pLV-07 (22). The vector designated as pLV-51 was introduced into the Agrobacterium tumefaciens strain LBA 4404 by the freezeand-thaw method.…”

“…The amplified fragment (830 bp) was treated with BglII and BamHI and fused with the BglII-predigested backbone, yielding an intermediary vector designated as pLV-60. Finally, the T-DNA part was cut out from the vector pLV-07 (22) as a BglII fragment and cloned into pLV-60. The resulting vector was designated as pLV-62 and contained the following fusion cassette: MB1 replication origin-BglII restriction site-right T-DNA border-multicloning site-nptII gene-left T-DNA border-BglII restriction site.…”

“…Petunia plants were cultivated in vitro on 1.0% agar containing an MS medium (25) under conditions described by Vrba and Matous ˇek (22). Plants were transformed according to a standard leaf disk method (26) and maintained on the medium containing 200 mg L -1 kanamycin and 200 mg L -1 timentin.…”

Isolation and characterization of a novel semi-lethal Arabidopsis thaliana mutant of gene for pentatricopeptide (PPR) repeat-containing protein

“…The 1.43 kb PCR product digested with ApaI and XbaI was cloned in between a cauliflower mosaic virus 35S promoter and a polyA signal within vector pLV-68, a derivative of pRT100 (Töpfer et al 1987). This PPR expression cassette was than cloned as an AscI-PacI restriction fragment into a binary vector pLV-07 (Vrba and Matoušek 2005) resulting in pLV-76. The intact gene was amplified using primers PPRwt5¢EcoRI (5¢-CGG AAT TCG TCC ATT ACA AAC CCT TC-3¢) and PPRwt3¢BglII (5¢-CCA TCT CAA GAT CTA CGC ACG-3¢) and the resultant 2.3 kb PCR product digested with EcoRI and BglII was ligated into EcoRI and BamHI digested vector pLV-07 resulting in binary vector pLV-77.…”