Martin Jopcik

Plant tissue-specific promoters can drive gene expression in Escherichia coli

Plant transgenesis often requires the use of tissue-specific promoters to drive the transgene expression exclusively in targeted tissues. Although the eukaryotic promoters are expected to stay silent in Escherichia coli, when the promoter-transgene units within the plant transformation vectors are constructed and propagated, some eukaryotic promoters have been reported to be active in prokaryotes. The potential activity of plant promoter in E. coli cells should be considered in cases of expression of proteins that are toxic for host cells, environmental risk assessment or the stability in E. coli of plant vectors for specific Cre/loxP applications. In this study, DNA fragments harbouring four embryo- and/or pollen-specific Arabidopsis thaliana promoters were investigated for their ability to drive heterologous gene expression in E. coli cells. For this, they were fused to gfp:gus reporter genes in the pCAMBIA1304 vector. Although BPROM, bacterial sigma70 promoter recognition program identified several sequences with characteristics similar to bacterial promoters including -10 and -35 sequences in each of tested fragments, the experimental approach showed that only one promoter fragment was able to drive relatively strong- and one promoter fragment relatively weak-GUS expression in E. coli cells. Remaining two tested promoters did not drive any transgene expression in bacteria. Our results also showed that cloning of a shorter plant promoter sequence into vectors containing lacZ α-complementation system can increase the probability of gene expression driven by upstream located lac promoter. This should be considered when cloning of plant expression units, the expression of which is unwanted in E. coli.

Field-based artificial crossings indicate partial compatibility of reciprocal crosses between Pinus sylvestris and Pinus mugo and unexpected chloroplast DNA inheritance

Crossability relationships between Scots pine (Pinus sylvestris L.) and mountain dwarf pine (Pinus mugo Turra) was studied, using artificial pollination approach. Partial compatibility of the reciprocal crossings of these species was proved experimentally, validating the idea of a spontaneous formation of their hybrid swarms under natural conditions. The hybrids were validated using organellar DNA markers and nuclear DNA microsatellites. Based on the percentage of filled seeds, the interspecific crossings were less efficient than the intraspecific cross-pollinations of P. sylvestris and P. mugo individuals. Both species were found to intercross readily with individuals of their putative hybrid swarm, P. mugo exhibiting a higher hybridological affinity towards putatively hybrid individuals than P. sylvestris. Validation of the hybrids confirmed the paternal inheritance of chloroplast DNA (cpDNA) in the combination P. sylvestris × P. mugo only. Surprisingly, in the reciprocal crossing P. mugo × P. sylvestris, maternal inheritance of cpDNA was revealed. Obtained results offer a new insight into the direction and intensity of gene flow within the hybrid swarms of Scots pine and mountain dwarf pine.

Optimalisation of expression conditions for production of round-leaf sundew chitinase (Drosera rotundifolia L.) in three E. coli expression strains

Round-leaf sundew (Drosera rotundifolia L.), family Droseraceae, genus Drosera, is one of a few plant species with a strong antifungal potential. Chitinases of carnivorous plants play an important role in decomposition of chitin-containing cell structures of insect prey. The cell wall of many phytopathogenic fungi also contains chitin, which can be utilized by chitinases, thus round-leaf sundew represents an interesting gene source for plant biotechnology. The purpose of this study was to compare the suitability of 3 different E. coli expression strains (E. coli BL21- CodonPlus® (DE3)-RIPL, E. coli ArcticExpress (DE3)RIL and E. coli SHuffle® T7) for production and isolation of heterologous round-leaf sundew chitinase (DrChit). Results showed that the recombinant protein was successfully expressed in all three strains, but occurred in insoluble protein fraction. To get the DrChit protein into soluble protein fraction some modifications concerning to induction temperatures and concentration of the IPTG inductor were tested. In addition, composition of lysis buffer has been modified with supplementation of strong non-ionic detergents, Triton® X100 and Tween® 20, respectively. As these modifications didn’t increase the amount of the DrChit protein in soluble fraction, therefore, its isolation under denaturing conditions and subsequent refolding for activity assays is recommended.

The expression profile of Arabidopsis thaliana β-1,3-glucanase promoter in tobacco

Well characterized promoters with specific activity only during male gametophyte development of transgenic organism are widely utilized in strategies aimed at the elimination of unwanted transgene escape from the transgenic to the non-transgenic plant population. Since the specificity and timing of the applied promoter in the original and transgenic organism don’t have to be consistent, here we have tested by promoter-GUS fusion analysis the APRS promoter isolated from Arabidopsis thaliana in transgenic tobacco plants. Unlike of A. thaliana transcriptomic microarray data that identified gene expression from this promoter in late stages of pollen development, in tobacco plants the APRS promoter was active in the developing microspores during a short period of time before the microspores are released from the tetrads. Despite these discrepancies, the APRS promoter remains strictly pollen specific in tobacco plants. However, verification of its specificity in other important crops should precede the use of the APRS promoter in the engineered male sterility or in production of transgene-free pollen via site-specific recombination systems.