Vectors construction

We have successfully constructed 2 backbones, piGEM001 and piGEM002. And we have verified them using digestion (Fig.1) and sequencing.

In order to enhance the ability of tobacco to metabolize formaldehyde, we have successfully constructed 3 multigenge expression vectors, from vector piGEM009 to vector piGEM011. We have verified all of them using digestion (Fig.3) and sequcencing.

Tobacco transformation

Tobacco was transformed essentially using the leaf disk co-cultivation protocol of Horsch. This protocol includes infection and co-cultivation (Fig.4A), selection (Fig.4B), regeneration and rooting (Fig.4C). We have successfully transformed each vector into babacco and got positive transgenic plantlets (Fig.5). Table 1 is the statistical result of quantity of each transgenic line. And we have got PCR positive plantlets of every transgenic line.

Expression of four key enzymes in tobacco

We extracted DNA from tobacco plants. And then we used specific primers to amplify the target gene to verify the kan-resistant plants (Fig.6). Next we extracted RNA from tobacco leaves which are PCR positive. We used RT-PCR to detect whether target gene was expressed (Fig.7).

Enhanced formaldehyde tolerance

The transgenic and wildtype plants, which had been grown separately in sealed boxes, were exposed to formaldehyde evaporated from a micro tube (0.5ml) containing formaldehyde solution (37%, 10ul) (Fig.8). One week later we observed the phenotype of transgeneic and widetype plants (Fig.9). We found that the transgenetic plant is stronger than wildtype after formaldehyde exposure. This indicates that production of HPS/PHI, FALDH and FDH enhanced formaldehyde tolerance of transgenic plants.

Enhanced formaldehyde absorbance

We detected the concentration of gaseous formaldehyde evaporated from a micro tube (0.5ml) containing formaldehyde solution (37%, 10ul) and made a curve (Fig.10) about relationship between formaldehyde concentration and time. And we saw a linear relationship between formaldehyde concentration and time before formaldehyde is saturated. For quantity result, we used a formaldehyde detector to detect the concentration of gaseous formaldehyde (Fig.11). The transgenic plants and wildtype, which had been grown separately in sealed boxes, were exposed to formaldehyde evaporated from a micro tube (0.5ml) containing formaldehyde solution (37%, 50ul) for about 2 weeks. Two weeks later, the covers of the plant boxes were removed and quickly replaced with covers equipped with formaldehyde dose-monitoring tubes in order to determine roughly the gaseous formaldehyde levels remaining in the boxes. We have not got the precise data results now, and this work is to be continued.

Transit peptides affect formaldehyde degrading efficiency

HPS, PHI, and FDH are located in chloroplast, while FALDH plays a role in cytoplasm. So we used transit peptides to locate the productions of these genes. We hope to know the effects of transit peptide on degrading formaldehyde. So we exposed transgenic tobaccos with and without transit peptides to formaldehyde (37%, 10ul). However, there are no obvious phenotype difference compairing different transgenic lines because time limited.

Different genes affect formaldehyde degrading efficiency

To test which enzymes play the most important role in pathway of metabolizing formaldehyde, we constructed mono-gene expression vectors to express each enzyme individually. We also constructed multi-gene expression vectors to test whether the ability of metabolizing formaldehyde of transgenic tobacco enhanced. Then these transgenic tobaccos were exposed to formaldehyde (37%, 10ul). However, we have not got obvious phenotype difference among transgenic lines, because there is not enough time

Tapetal expression of AdCP results in male sterility in high expression plants

Considering the problem of environment and safety, we use male sterility system which prevents the horizontal transgene flow. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion. However, there is not enough time to wait for transgenic tobacco flowering.