Key parts

Fig.1 A diagrammatic sketch about our key parts.

FALDH (BBa_K1537026)

The glutathione-dependent formaldehyde dehydrogenase (FALDH) plays a key role in formaldehyde metabolism. FALDH is identified as an enzyme expressed in the cytoplasm. If we make FALDH over-express in plants, we can enhance plants’ tolerance to formaldehyde and increase the ability of plants to absorb formaldehyde. In the process of metabolism of formaldehyde, the formaldehyde may first combine with glutathione (GSH) to form the product of S-hydroxymethyl glutathione (HM-GSH), then FALDH in cytoplasm will catalyzes the formation of a S-formyl glutathione (F-GSH). Next the F-GSH will be hydrolyzed to formate (HCOOH) and GSH by S-formyl glutathione hydrolase (FGH).

FDH (BBa_K1537025)

Formate dehydrogenase (FDH) is a mitochondrial-localized NAD-requiring enzyme while the HCOOH is getting into the mitochondrial, FDH will oxidize the formic acid into CO2, and reduce NAD+ to NADH with a high degree of specificity. In our project, the heterologous expression of FDH from arabidopsis thaliana was completed.

HPS and PHI (BBa_K1537024)

The ribulose monophosphate (RuMP) pathway is one of the formaldehyde-fixation pathways found in microorganisms called methylotrophs, which utilize one-carbon compounds as the sole carbon source. The key enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS), which fixes formaldehyde to D-ribulose-5-phosphate (Ru5P) to produce D-arabino-3-hexulose-6-phosphate (Hu6P), and 6-phospho-3-Hexuloiso-merase (PHI), which converts Hu6P to fructose 6-phosphate (F6P). The two key enzymes work in chloroplast both. We will use fusion expression to conduct heterologous expression in tobacco.

Part for safety

AdCP (BBa_K1537027)

Considering the problem of environment and safety, we use male sterility system which prevents the horizontal transgene flow. Pawan Shukla has used a plant pathogen-induced gene, cysteine protease to induce male sterility. This gene was identified in the wild peanut, Arachis diogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsis personata. Arachis diogoi cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29). And tobacco transformants were generated. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion.

Part for stomatal expand

AHA2 (BBa_K1537028)

Stomata are microscopic pores surrounded by two guard cells and play an important role in the uptake of CO2 for photosynthesis. Recent researches revealed that light-induced stomatal opening is mediated by at least three key components: blue light receptor phototropin, plasma membrane H+-ATPase, and plasma membrane inward-rectifying K+ channels. However, Yin Wang, et al, showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening. Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plantgrowth. Therefore, in order to improve the ability of absorbing formaldehyde, we overexpresse H+-ATPase (AtAHA2) in transgenic tobacco guard cells , resulting in a significant effect on light-induced stomatal opening.

Promoters

35S promoter (BBa_K1537015)

The 35S promoter is a strong promoter derived from cauliflower mosaic virus. This constitutive promoter is widely used in transgenic plants to improve the level of the expression of foreign genes effectively.

TA29 promoter (BBa_K1537019)

TA29 promoter is a tissue-specific (tapetal cells) promoter found in tobacco.

GC1 promoter (BBa_K1537020)

The GC1 promoter drives strong reporter expression in guard cells of Arabidopsis and tobacco plants. It provides a potent research tool for targeted guard cell expression.

Terminators

HSP terminator (BBa_K1537029)

The heat shock protein 18.2 (HSP) terminator was the most effective in supporting increased levels of expression. The HSP terminator increases mRNA levels of both transiently and stably expressed transgenes approximately 2-fold more than the NOS (nopaline synthase) terminator in transfected Arabidopsis T87 protoplasts. When combined with the HSP terminator, a translational enhancer increased gene expression levels approximately 60- to 100-fold in transgenic plants.

CaMV35S polyA (BBa_K1537029)

It’s a kind of terminator derived from cauliflower mosaic virus.

NOS terminator (BBa_K1537031)

It’s quite a common terminator in expression system of plants.

Transit peptides

TCP01 (BBa_K1537021)

TCP02 (BBa_K1537022)

They both are chloroplast transit peptides. We use them to guide HPS-PHI and FALDH into chloroplast.

TCP03 (BBa_K1537023)

TCP03 is also a kind of transit peptide which can lead formate dehydrogenase into chloroplast.

Improved Parts

35S promoter + translation initiation optimized sequence for dicot + MASS translation enhancer(BBa_K1537015)

Pre-existing Part:BBa_K414002

Our 35S promoter is based on BBa_K414002, and we also add translation initiation optimized sequence for dicot and MASS translation enhancer after 35S promoter to enhance gene-expression in plants.

GSG linker+P2A (BBa_K1537016)

GSG linker+T2A (BBa_K1537017)

Pre-existing Part: BBa_K1199016, BBa_K1199046

We added GSG linker to enhance cleavage. In addition, we use 3 kinds of 2As rather than only one 2A in a vetor.

GSG linker is an oligopeptide of “Gly-Ser-Gly” between your protein and 2A peptide to enhance cleavage.
The 18~22 amino acids 2A self-cleaving oligopeptides can be used for co-expression of multiple, discrete proteins from a single ORF. Based on highly inefficient peptide bond formation between glycine and proline residues within the 2A peptide, placement of 2A peptide sequence as a linker region between tandem cDNA’s allows the stoichiometric translation of multiple unfused protein products. To minimize the risk of homologous recombination, it is important to use different 2A peptide sequences if more than two genes are being linked. The 2A peptide system has thus far worked successfully in all eukaryotic systems tested, from mammalian cells, yeast, and plants. In our project,we use F2A , P2A and T2A to achieve our goal.