我们参赛的项目旨在通过植物吸收代谢甲醛，达到降低室内甲醛浓度的目的。我们通过将微生物和植物中代谢甲醛的关键酶基因3-hexulose-6-phosphate（HPS）、6-phospho-3-hexuloisomerase(PHI)、formaldehyde dehydrogenase(Faldh)、?formate dehydrogenase(FDH）、气孔调节基因（At AHA2），利用基因工程的手段在模式植物烟草中过量表达，最终达到加强植物对室内甲醛降解能力的目的。考虑到生物安全性，我们还将花粉败育基因（Ad CP）在烟草中过量表达，阻止花粉传播造成的基因污染。另外，我们还计划将微生物中代谢甲醛的关键酶基因转到烟草的叶绿体基因组中，保证生物安全性。

Stomatal opening

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[1]showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening（Fig. 1）. Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plant growth. Therefore,in order to improve the ability of absorbing formaldehyde, we overexpresse H+-ATPase(At AHA2) in transgenic tobacco guard cells ,resulting in a significant effect on light-induced stomatal opening.


Fig.1 Typical stomata in the epidermis illuminated with light for 30 min[1].

Biosafty

In order to improve biology safety,we use male sterility systems which can be used as a biological safety containment to prevent horizontal transgene flow. Pawan Shukla[2] has used a plant pathogen-induced gene, cysteine protease for inducing 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.


Fig. 2 In vitro pollen germination of untransformed control plant and sterile transgenic plants. Pollen grains were germinated on sucrose-boric acid medium and >500 pollen grains were observed.a Untransformed control plant pollen, b Sterile pollen.Scale bar 25 μm[2]

Vectors

In order to measure the ability of different genes in the metabolism of formaldehyde ,We constructed 11 gene expression vectors（Table 1 and Fig. 3），including 2 backbone vectors，6 single gene expression vectors and 3 multi-gene expression vectors.


piGEM001: P35S+P2A+T2A+F2A+nptII+T-HSP+T-35S+T-nos
piGEM002: piGEM001+AtAHA2+PGC1+PTA29+AdCP
piGEM003: piGEM002+TCP02-HPS-PHI
piGEM004: piGEM002+TCP03-AtFDH
piGEM005: piGEM002+TCP01-BbFALDH
piGEM006: piGEM002+HPS-PHI
piGEM007: piGEM002+AtFDH
piGEM008: piGEM002+BbFALDH
piGEM009: piGEM002+HPS-PHI+AtFDH+BbFALDH
piGEM010: piGEM002+TCP02-HPS-PHI+TCP03-AtFDH+FALDH
piGEM011: piGEM002+TCP02-HPS-PHI+TCP03-AtFDH+TCP01-BbFALDH
piGEM012: piGEM001+TCP02-HPS-PHI+TCP03-AtFDH+TCP01-BbFALDH

2A peptide Sequence[3-6

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


Fig. 4 schematic of 2A peptide
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. These sequences were first discovered in the foot-and-mouth disease virus (FMDV).And since than 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 Thosea asigna virus) to achieve our goal.

Gibson Assemble

In 2009 Dr. Daniel Gibson and colleagues at the J. Craig Venter Institute developed a novel method for the easy assembly of multiple linear DNA fragments (Nat Methods 2009;6(5):343-5).（Fig. 5） Regardless of fragment length or end compatibility, multiple overlapping DNA fragments can be joined in a single isothermal reaction. With the activities of three different enzymes, the product of a Gibson Assembly is a fully ligated double-stranded DNA molecule. This has proven to be an efficient and effective method for the assembly of plasmids, and molecular biologists now use this method extensively


Fig. 5 schematic of Gibson Assemble
The Gibson Cloning Master Mix consists of three different enzymes within a single buffer. Each enzyme has a specific and unique function for the reaction:
T5 Exonuclease?-creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each other.
Phusion DNA Polymerase?-incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments.
Taq DNA Ligase?-covalently joins the annealed complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment.

Reference

[1] Yin Wang,et al.(2014) Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatalopening and enhances plant growth. 111(1):533-8. doi: 10.1073/pnas.1305438111
[2]Pawan Shukla et al. Expression of a pathogen-induced cysteine protease (AdCP) in tapetum results in male sterility in transgenic tobacco Funct Integr Genomics DOI 10.1007/s10142-014-0367-2
[3][3]Andrea?L.?Szymczak-Workman,?Kate?M.?Vignali,?and?Dario?A.A.?Vignali.Design?and?construction?of?2A?vectors.Cold?Spring?Harb?Protoc?2012;doi:10.1101/pdb.ip067876
T5 Exonuclease?-creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each other.
[4]Abdelhak?El?Amrani,Abdellah?Barakate,Barak?M.?Askari,?Xuejun?Li,?Alison?G.?Roberts,Martin?D.?Ryan,?and?Claire?Halpin.Coordinate?Expression?and?Independent?Subcellular?Targeting?of?Multiple?Proteins?from?a?Single?Transgene.Plant?Physiology,?May?2004,?Vol.?135,?pp.?16–24
[5] Pablo?de?Felipe,?Lorraine?E.?Hughes,?Martin?D.?Ryan,?and?Jeremy?D.?Brown.Co-translational,?Intraribosomal?Cleavage?of?Polypeptides?by?the?Foot-and-mouth?Disease?Virus?2A?Peptide.J.?Biol.?Chem.?2003,?278:11441-11448
[6]Andrea?L?Szymczak,?Creg?J?Workman1,Yao?Wang,?Kate?M?Vignali,?Smaroula?Dilioglou,?Elio?F?Vanin&?Dario?A?A?Vignali.?Correction?of?multi-gene?deficiency?in?vivo?using?a?single?‘self-cleaving’?2A?peptide–based?retroviral?vector.Nature?Biotechnology?22,?589?-?594?(2004)