High transformation frequency of tobacco and rice via Agrobacterium‐mediated gene transfer by flanking a tobacco matrix attachment region

Abstract

Nuclear matrix attachment regions (MARs) are suggested to regulate chromatin structure and influence the expression of flanking genes. Our previous study showed that TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro and increase transgene expression in vivo. Here, we investigated the role of TM2 MAR in improving transformation frequency of Agrobacterium‐mediated transformation in tobacco and rice. The gusA reporter gene flanked by TM2 MAR in pBI121 and pCAMBIA‐1301 vectors was controlled by a constitutive promoter or a photosynthetic tissue‐specific promoter. The presence of TM2 MAR in different expression cassettes significantly increased the numbers of kanamycin‐resistant tobacco shoots, hygromycin‐resistant rice calli and shoots. Seeds from the independent transgenic lines with TM2 MAR can germinate normally on the medium containing 500 mg l−1 kanamycin, whereas none of the seeds from the transgenic lines without TM2 MAR survived. Furthermore, RNA gel blot analysis revealed that nptII messenger RNA levels are more abundant in the independent transgenic lines with TM2 MAR than in the lines without TM2 MAR. Altogether, these data reveal a possible mechanism that TM2 MAR improves the transformation frequency by increasing nptII gene expression.

Functional characterization of a tobacco matrix attachment region-mediated enhancement of transgene expression

“…This result is different from that found for another tobacco MAR in the 5 0 -region of CHN50 (Fukuda and Nishikawa 2003); however, we did find that the 3 0 -MAR exhibited a weaker effect than the 5 0 MAR. These results suggest that TM2 acts in a bidirectional manner, which is consistent with the improvement in transformation frequency by highlevel expression of the nptII gene located at the upstream of the 5 0 -MAR (Zhang et al 2007). Based on results on different mini-promoters, the 5 0 -MAR did not increase the expression of the gusA gene derived by the 35S mini-promoter, which is consistent with results reported for the 5 0 -MAR of the CHN50 gene and b-phaseolin gene (van der Geest and Hall 1997;Fukuda and Nishikawa 2003).…”

“…The sterile water-rinsed leaves were cut into 5-mm 2 pieces. The constructed chimeric plasmid constructs were transferred into Agrobacterium tumefaciens strain LBA4404 and used to transform the leaf pieces as previously described (Zhang et al 2007). The transfected leaf pieces were subcultured on regeneration medium consisting of MS salts (Murashige and Skoog 1962) supplemented with promoter (P35S)-gusA-nos terminator (Tnos) cassette are labeled as cassettes I, II and III, respectively.…”

“…This MAR does not change the gene expression pattern controlled by the CaMV 35S constitutive promoter and PNZIP photosynthetic tissue-specific promoter (Xue et al 2005), but it can improve the transformation frequency so as to decrease gene silencing in transformants by flanking the TM2 on both sides of the transgene expression cassette (Zhang et al 2007). These results suggest that this MAR has a great potential in terms of facilitating genetic improvements in a wide range of plant species.…”

Plastid targeting strategies for cyanophycin synthetase to achieve high‐level polymer accumulation in Nicotiana tabacum

“…In accord with previously published data (Zhang et al ., 2007), a correlation was observed between the transcript level and cyanophycin content in descendants of PsbY‐ cph A Te line 51 (Figure 4b): plant 51‐3‐2, with a higher transcript level of cph A, also showed an increased polymer accumulation of up to 68.42 mg/g dry weight, relative to 51‐5‐18 (7.96 mg/g dry weight).…”

NtKTI1, a Kunitz trypsin inhibitor with antifungal activity fromNicotiana tabacum, plays an important role in tobacco’s defense response

“…Nc89 was grown on soil until the six-leaf stage. The fusion gene constructs were transferred to Agrobacterium tumefaciens strain LBA4404 by the freeze-thaw method and the leaf pieces were transformed as described previously [56]. The pBI121-TM2 empty vector was transformed as a control.…”