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 HCHO and increase the ability of plants to absorb HCHO. In the process of metabolism of formaldehyde, the formaldehyde may first combined 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 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 HCHO-fixation pathways found in microorganisms called methylotrophs, which utilize one-carbon compoundsas the sole carbon source. The key enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS), which fixes HCHO 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 conductheterologous 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 cellsand play an important role in the uptake of CO2 for photosynthesis.Recent researches revealed that light-induced stomatalopening 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[1]showed that only increasing the amount of H+-ATPase in guardcells had a significant effect on light-induced stomatal opening.Transgenic Arabidopsis plants by overexpressing H+-ATPase inguard cells exhibited enhanced photosynthesis activity and plantgrowth. Therefore,in order to improve the ability of absorbingformaldehyde, we overexpresse H+-ATPase(At AHA2) 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 terminatorderived from cauliflower mosaic virus.

NOS terminator(BBa_K1537031)

It’s quite a commonterminator in expression system of plants.

Transit peptides

TCP01(BBa_K1537021)

TCP02(BBa_K1537022)

It’s chloroplast transit peptides. We use them to guide HPS-PHI and FALDH into chloroplast.

TCP03(BBa_K1537023)

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

Improved Parts

35S promoter(BBa_K1537015)

We added translation initiation optimized sequence for dicot and mass translation enhancer after 35S promoter to enhance gene-expression in plants. More details for previous parts

GSG linker+P2A(BBa_K1537016)

GSG linker+T2A(BBa_K1537017)

We added GSG linker to enhance cleavage. In addition, we use 3 kinds of 2A 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 ofmultiple, discrete proteins from a single ORF(Fig.1)Based onhighly inefficient peptide bond formation between glycineand proline residues within the 2A peptide, placementof 2A peptide sequence as a linker region betweentandem cDNA’s allows the stoichiometric translation ofmultiple unfused protein products. To minimize therisk of homologous recombination, it is important to use different 2A peptide sequences if morethan two genes are being linked.The 2A peptide system has thus far worked successfully in all eukaryotic systems tested, from mammaliancells, yeast, and plants.In our project,we use F2A(from foot-and-mouth disease virus), P2A(from porcine teschovirus-1) and T2A(from Thosea asigna virus) to achieve our goal.