Vectors Construction

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

Fig. 1 Digestion the plasmid piGEM001 with HpaI and SpeI
M: DNA molecular weight marker
2: piGEM001_2(false positive)
3: piGEM001_3

Fig. 2 Digestion the plasmid piGEM002 with HpaI and DraIII
M: DNA marker
4: piGEM002_4 and product of digestion
5: piGEM002_5 and product of digestion
7: piGEM002_7 and product of digestion
8: piGEM002_8 and product of digestion

Then we have successfully constructed 6 monogene expression vectors, from vector piGEM003 to vector piGEM008. And we have verified all of them using digestion and sequcencing. Here we only show the result of vector piGEM005(Fig. 3). You can browse notebook for more results.

Fig. 3 Digestion the plasmid piGEM005 with EcoRI and SacI.
M: DNA marker
5: piGEM005 plasmid and TCP02-HPS-PHI fragment

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.4, Fig.5 and Fig.6) and sequcencing.

Plant transformation

Tobacco was transformed essentially using the leaf disk co-cultivation protocol of Horsch.This protocol includes three stages, co-cultivation (Fig. 7), screening cultivation (Fig. 8) and rooting cultivation (Fig.9). We have successfully transformed each vector into babacco

Fig. 7 Transform piGEM003、piGEM004、piGEM005、 piGEM006、 piGEM007、 piGEM008、 piGEM009、 piGEM010、 piGEM011 into tobacco, co-cultured for 48 hours.

Fig. 8 First round to filtrate the positive clone from piGEM003、piGEM004、piGEM005、 piGEM006、 piGEM007、 piGEM008、 piGEM009、 piGEM010、 piGEM011 for 2 weeks. Filter pressure: 50mg/L kanamycin.

Fig. 9 Transform piGEM003、piGEM004、piGEM005、 piGEM006、 piGEM007、 piGEM008、 piGEM009、 piGEM010、 piGEM011 into tobacco, and let it into root culture.Filter pressure: 50mg/L kanamycin.

Molecular identifition

We extracted DNA from mature tobacco seedlings. And then we used specific primers to amplify the target gene to verify the positive seedlings (Fig. 10). Next we extracted RNA from tobacco leaves which are DNA positive.we used RT-PCR to detect whether target gene was expressed.

Fig. 10 PCR identification of piGEM004-transformed positive tobacco seedlings.
M: DNA marker
H2O: negative control
WT: widetype control
P: positive control
1-11: 11 different lines

Phenotype testing

Finally we expose the tobacco seedlings into formaldehyde surroundings. One week later We observed the phenotype of transgeneic plants and widetype (Fig. 12). We found that the transgenetic seedling is stronger than wildtype after formaldehyde exposure.

Fig. 12 Phenotype testing of transgenetic seedlings and wildtype. A. Before exposure to HCHO. B. Exposure to HCHO for one week.The transgenetic seedling is stronger than wildtype after formaldehyde exposure. 20ul 37% HCHO , one week.

For quantity result, we used a HCHO detector to detect the concentration of gaseous HCHO (Fig. 13).

Fig.13 Gaseous HCHO tolerance and uptake capacity of transgenic tobacco plants(Li-Men Chen et, al. 2009)

These results indicate that the HCHO assimilation pathway strongly enhanced not only the tolerance of the transgenic plants to exogenous HCHO, but also their ability to take up and eliminate gaseous HCHO.