GSG linker

This part of GSG is used by improving the previous work from other iGEM team.

GSG linker is anoligo-peptide of “Gly-Ser-Gly” between your protein and 2A peptide to enhance cleavage. Ribosomal “skipping” is an alternate mechanism of translation in which a specific viral peptide prevents the ribosome from covalently linking a new inserted amino acid, and let it continue translation. This results in apparently co-translational cleavage of the poly-protein.

This process is induced by a “2A-like”, or CHYSEL (cis-acting hydrolase element) sequence. This sequence comprises a non-conserved sequence of amino-acids with a strong alpha-helical propensity followed by the consensus sequence -D(V/I)ExNPG P, where x= any amino acid. The apparent cleavage occurs between G and P.

Ribosomal “skipping” has been observed only in +ssRNA and dsRNA viruses, whose hosts are animals, plants or insects. The conserved motif has been identified also in trypanosoma and in some fungus proteins. Ribosomal “skipping” is functioning both in vitro and in vivo when translated by eukaroytic ribosomes, but is inactive when translated by prokaryoticribosomes.

35S promoter

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.

Mass translation enhancer

Insertion of “GCT TCCTCC” after the initiator codon ATG can augmentdownstreamgene-expression in plants.

2A

2A peptide sequenceswere found in Picornaviruses to mediate "cleavage" between two proteins.We use 2A peptide-linked multi-cistronic vectors to express multiple proteins from a single open reading frame (ORF)effectively.

The 18-22 amino acids 2A self-cleaving oligo peptides 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. These sequences were first discovered in the foot-and-mouth disease virus (FMDV). And since then many 2A-like sequences have been identified in other viruses and some parasites. 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 (from foot-and-mouth disease virus), P2A (from porcine teschovirus-1) and T2A (from Thoseaasigna virus) to achieve our goal.

TA29 promoter

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

GC1 promoter

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.

HPS and PHI

The ribulose monophosphate (RuMP) pathway is one of the HCHO-fixation pathways found in microorganisms called methylotrophs, which utilizes 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-hexuloisomerase (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. The bacterial RuMP pathway and the plant Calvin-Benson cycle share common metabolic features: (i) bothpathways fix a one-carbon unit to ribulose phosphate;(ii) the fixation reaction eventually yields F6P; and (iii)Ru5P is regenerated from F6P through rearrangementreactions.In the pathwaydesigned, the CO2-fixation and reduction steps in thecycle are bypassed by the HPS- and PHI-catalyzedreactions.

HPS and PHI denote 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase respectively. The abbreviations for several sugarphosphates are as follows: Ru5P, ribulose 5-phosphate; Hu6P, hexulose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate;RuBP,ribulose1,5-bisphosphate;3-PGA,3-phosphoglyce-rate. The other metabolites in the pathway are symbolized merely by their carbonnumbers for simplicity.

FDH

Formate dehydrogenase is a mitochondrial-localized NAD-requiring enzyme while HCOOH is getting into 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.

The abbreviationsare as follows:
CAT:catalase;
FALDH:glutathione-dependent formaldehyde dehydrogenase;
FGH:S-formylglutathione hydrolase;
FDH: Formate dehydrogenase;
SYN: 10-Formyl-THFsynthetase;
FTD: 10-formyltetrahydrofolatedehydrogenase;
MTD: 5,10-methylenetetrahydrofolate dehydrogenase;
MTC:5,10-methylenetetrahydrofolate cyclohydrolase;
SHMT: Serine hydroxymethyl transferase;
GDC: Glycine decarboxylase complex;
GXS: Glyoxalic acid synthetase;
GXDC: Glyoxalic acid decarboxylase;
HM-GSH: S-Hydroxymethyl glutathione;
Forml-GSH: Formyl glutathione;
SMM cycle: Methionine cycle.Zhang Wei.The studies on formaldehyde metabolicmechanism in Petunia hybrida and the geneticmanipulation for enhancing its formaldehydephytoremediation ability.A dissertation submitted for the degree ofMaster of Scienceat theKUNMING UNIVERSITY OF SCIENCE ANDTECHNOLOGY (2012).

FALDH

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).
In paper “Overexpression of the Formaldehyde DehydrogenaseGene from Brevibacillus brevis to Enhance FormaldehydeTolerance and Detoxification of Tobacco”,the gaseous H13CHO metabolic spectrum in the transgenic and WT tobacco wasanalyzed using 13C-NMR.

Fig.6: a C13-NMRspectrafrom leaf extracts of transgenic tobacco plant treated with gaseousH13 CHO for 2 hour; b C13-NMR spectrafrom leaf extracts of WT (Wild Type) treated with gaseous H13 CHO for 2 hour; c The extract from WT (Wild Type) plant leaves without H13 CHO treatment was used to monitor the background C13-NMR signal levels.

AdCP

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, Arachisdiogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsispersonata. Arachisdiogoi 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.

Fig.7 Comparison of flowermorphology of male sterile T2transgenic plants(a) and fully openednon-transformed control plant flower (c);Fully opened, flowerof non-transformed controlflower (b, d). Stamen length hasbeen reduced in male steriletransgenic plants (e) compared tothe non-transformed control plant(f).

Fig. 8: Pollen characteristics ofmale sterile transformed anduntransformed control plants. aand dare Alexander stain images,b,c, e, and f are SEM images. a,b, c Untransformed control plantpollen, d, e, f sterile pollen. Scalebar 25 μm.

AHA2

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 absorbing formaldehyde, we overexpress H+-ATPase(At AHA2) in transgenic tobacco guard cells,resulting in a significant effect on light-induced stomatal opening.

Fig. 11: (A) Typical fluorescence images of immunohistochemical detectionof the guard cell H+-ATPase in the Arabidopsis epidermis. (B) qPCR analysis of AHA2 expression. Error bars represent the SEM (n ≥ 6).Significant differences were detected by Student t test (*** P < 0.001). (C) Typical stomata in the epidermis illuminated with light for 30 minutes.

RBCS-3C

It’s chloroplast transit peptides.

TCP03

TCP03 isalso a kind of transiting peptide which can lead formate dehydrogenase into chloroplast.

HSP terminator

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

It’s a kind of terminatorderived from cauliflower mosaic virus.

NOS terminator

It’s quite acommonterminator in expression system of plants.