Overexpressions of dihydroxyacetone synthase and dihydroxyacetone kinase in chloroplasts install a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco using modified Gateway entry vectors

• Autorzy: Xiao S.-Q. , Sun Z. , Wang S.-S. , Zhang J. , Li K.-Z. , Chen L.-M.
• Języki publikacji: EN

Abstrakty

EN

Dihydroxyacetone synthase (DAS) and dihydroxyacetone kinase (DAK) are two key enzymes for formaldehyde assimilation in methylotrophic yeasts. In order to using a Gateway LR recombination reaction to construct a plant expression vector that contains the expression cassettes for the das and dak genes and allow the proteins encoded by the two target genes to be localized to the chloroplasts of transgenic plants, the entry vector pEN-L4*-PrbcS-*T-gfp-L3* contained the tomato rbcS 3C promoter (PrbcS) with its transit peptide sequence (*T) and a GFP reporter gene (gfp) was constructed in this study. To verify the applicability of pEN-L4*-PrbcS-*T-gfp-L3*, we generated an entry vector for the dak gene by replacing the gfp gene in this entry vector with the dak gene. We also generated an entry vector for the das gene by replacing the gus gene in another entry vector (pENTR*-PrbcS-*T-gus) with the das gene. Using these entry vectors and pK7m34GW2-8m21GW3, we successfully constructed the pKm-35S-PrbcS-*T-gfp-PROLD-PrbcS-*T-gus and the pKm-35S-PrbcS-*T-dak-PROLD-PrbcS-*T-das expression vectors. Our results showed that high expression of GUS was achieved in leaves, and the expressed GFP, DAS and DAK proteins could be targeted to the chloroplasts after the two expression vectors were used to transform tobacco. The overexpressions of DAS and DAK in the chloroplasts successfully created a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco. By utilizing these expression vectors, we not only successfully expressed two target genes with one transformation but also localized the expressed proteins to chloroplasts via the transit peptide sequence (*T). Therefore, the construction of pEN-L4*-PrbcS-*T-gfp-L3* establishes a technique platform that provides a convenient means for chloroplast genetic engineering.

• Wydawca: -
• Czasopismo: Acta Physiologiae Plantarum
• Rocznik: 2012
• Tom: 34
• Numer: 5
• Opis fizyczny: p.1975-1985,fig.,ref.

Twórcy

autor

Xiao S.-Q.

• Faculty of Environmental Science and Engineering, Chenggong Campus, Kunming University of Science and Technology, 650500 Chenggong, Kunming, China
• Faculty of Life Science and Biotechnology, Chenggong Campus, Kunming University of Science and Technology, 650500 Chenggong, Kunming, China

autor

Sun Z.

• Faculty of Life Science and Biotechnology, Chenggong Campus, Kunming University of Science and Technology, 650500 Chenggong, Kunming, China

autor

Wang S.-S.

• Faculty of Life Science and Biotechnology, Chenggong Campus, Kunming University of Science and Technology, 650500 Chenggong, Kunming, China

autor

Zhang J.

• Faculty of Life Science and Biotechnology, Chenggong Campus, Kunming University of Science and Technology, 650500 Chenggong, Kunming, China

autor

Li K.-Z.

• Faculty of Life Science and Biotechnology, Chenggong Campus, Kunming University of Science and Technology, 650500 Chenggong, Kunming, China

autor

Chen L.-M.

• Faculty of Life Science and Biotechnology, Chenggong Campus, Kunming University of Science and Technology, 650500 Chenggong, Kunming, China

Bibliografia

• Chen LM, Yurimoto H, Li KZ, Orita I, Akita M, Kato N, Sakai Y, Izui K (2010) Assimilation of formaldehyde in transgenic plants due to the introduction of the bacterial ribulose monophosphate pathway genes. Biosci Biotechnol Bioch 74:627–635
• Chung SM, Frankman EL, Tzfira T (2005) A versatile vector system for multiple gene expression in plants. Trends Plant Sci 10:357–361
• Goderis IJ, De Bolle MF, François IE, Wouters PF, Broekaert WF, Cammue BP (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol 50:17–27
• Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
• Karimi M, Depicker A, Hilson P (2007) Recombinational cloning with plant gateway vectors. Plant Physiol 145:1144–1154
• Landy A (1989) Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem 58:913–949
• Lin L, Liu YG, Xu XP, Li BJ (2003) Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proc Natl Acad Sci USA 100:5962–5967
• Ma L, Song ZB, Hu QQ, Zhao Y, Nian HJ, Yu YX, Li KZ, Chen LM (2011) Construction and application of a Gateway entry vector with rubisco small subunit promoter and its transit peptide sequence*. Prog Biochem Biophys 38:269–279
• Miyagawa Y, Tamoi M, Shigeoka S (2001) Overexpression of a cyanobacterial fructose-1, 6-sedoheptulose-1, 7-bisphosphatase in tobacco enhances photosynthesis and growth. Nat Biotechnol 19:965–969
• Schmitz H, Hilgers U, Weidner M (2000) Assimilation and metabolism of formaldehyde by leaves appear unlikely to be of value for indoor air purification. New Phytol 147:307–315
• Song ZB, Orita I, Yin F, Yurimoto H, Kato N, Sakai Y, Izui K, Li KZ, Chen LM (2010) Overexpression of an HPS/PHI fusion enzyme from Mycobacterium gastri in chloroplasts of geranium enhances its ability to assimilate and phytoremediate formaldehyde. Biotechnol Lett 32:1541–1548
• Tzfira T, Tian GW, Lacroix B, Vyas S, Li J, Leitner-Dagan Y, Krichevsky A, Taylor T, Vainstein A, Citovsky V (2005) pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants. Plant Mol Biol 57:503–516
• Yurimoto H, Kato N, Sakai Y (2005) Assimilation, dissimilation, and detoxification of formaldehyde, a central metabolic intermediate of methylotrophic metabolism. Chem Rec 5:367–375
• Zhao Y, Christensen SK, Fankhauser C, Cashman JR, Cohen JD, Weigel D, Chory J (2001) A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science 291:306–309

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