Plasmid Construction

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

Fig.1 Verification of backbones using digestion
A. Digestion the plasmid piGEM001 with HpaI and SpeI. M: DNA marker; 1: plasmid piGEM001 and its digestion product B. Digestion the plasmid piGEM002 with HpaI and DraIII. M: DNA marker; 1: plasmid piGEM002 and its digestion production

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.2). You can browse notebook for more results.

Fig.2 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.3) and sequcencing.

Fig.3 Verification of multi-gene expression vectors using digestion A.Digestion the plasmid piGEM009 with XbaI and SalI. M: DNA marker; 1: piGEM009 plasmid and it's digestion product B. Digestion the plasmid piGEM010 with HindIII and SacI. M: DNA marker; 1: piGEM010 plasmid and it's digestion product C. Digestion of the plasmid piGEM011 with EcoRI and SacI. M: DNA marker; 6: plasmid piGEM011 and it's digestion production

Plant transformation

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

Fig.4 Transform piGEM003, piGEM004, piGEM005, piGEM006, piGEM007, piGEM008, piGEM009, piGEM010 and piGEM011 into tobacco. Co-cultured for 48 hours(A); Screening cultivation for one month(B and C); Rooting cultivation for one month (D).

Fig.5 positive tobacco seedlings of each transgenic lines.

Table.1 Statistical result of quantity of each transgenic line.

* Rate of Kan-resistant plantlet = N0.of Kan-resistant plantlet / No. of transformed tobacco Leaf disc
**Rate of positive seedlings =No. of PCR positive plantlet / N0.of Kan-resistant plantlet

Expression of four key enzymes in tobacco

We extracted DNA from tobacco plantlets. And then we used specific primers to amplify the target gene to verify the kan-resistant seedlings (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).

Fig.6 PCR identification of kan-resistant tobacco seedlings (piGEM010 transgenic line). M: DNA marker; WT: widetype control; P: plasmid; 1-8: 6 individual lines

Fig.7 RT-PCR verification of positive transgenic tobacco seedlings.
M: DNA marker; 1-8: 6 individual lines; 9: plasmid

Enhanced HCHO Tolerance

The transgenic plants and wildtype, which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%, 50ul) (Fig.8). One week later we observed the phenotype of transgeneic plants and widetype (Fig.9). We found that the transgenetic seedling is stronger than wildtype after formaldehyde exposure.This indicates that production of HPS/PHI, Faldh and FDH enhanced HCHO tolerance of transgenic seedlings to some extent.

Fig.8 The transgenic plants (A) and wildtype (B), which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%,50ul)

Fig.9 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.

Enhanced HCHO Absorbing

We detected the concentration of gaseous formaldehyde evaporated from a micro tube (0.5ml) containing HCHO solution (37%,10ul) and made standard curve (Fig.9). And we saw a linear relationship between HCHO concentration and time before formaldehyde is saturated. For quantity result, we used a HCHO detector to detect the concentration of gaseous HCHO (Fig.9). The transgenic plants and wildtype, which had been grown separately in sealed boxes, were exposed to HCHO evaporated from a micro tube (0.5ml) containing HCHO solution (37%,50ul) for about 2 weeks (Fig.9). Two weeks later, the covers of the plant boxes were removed and quickly replaced with covers equipped with HCHO dose-monitoring tubes in order to determine roughly the gaseous HCHO levels remaining in the boxes. As in the Fig.9, the concentrations were found to be less than 20 ppm for transgenic lines, but more than 20 ppm for wildtype plants. 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.

Fig.9 Standard Curve of Formaldehyde.

Fig.10 folmaldehyde Concentration Detection.

Transit Peptides Affect HCHO 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 HCHO. So we exposed transgenic tobaccos with and without transit peptides to HCHO (37%, 50ul). However, there are no obvious phenotype difference compairing different transgenic lines because time limited.

Different Genes Affect HCHO Degrading Efficiency

To test which enzymes play the most important role in pathway of metabolizing HCHO, 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 HCHO of transgenic tobacco enhanced. Then these transgenic tobaccos were exposed to HCHO (37%, 50ul). 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 until transgenic tobacco flowers.