pMAA-Red: a new pPZP-derived vector for fast visual screening of transgenic Arabidopsis plants at the seed stage

BMC Biotechnology

Muhammad Amjad Ali²  Kausar Hussain Shah²  Holger Bohlmann¹ 

[1] Division of Plant Protection, Department of Crop Sciences, University of Natural Resources and Life Sciences, UFT Tulln, Konrad Lorenz Strasse 24, 3430, Tulln, Austria

[2] Division of Plant Protection, Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Austria

关键词: Arabidopsis transformation;    Agrobacterium;    DsRed;    Agroinfiltration;    Marker gene;    pPZP family vectors;    Transient expression;   

Others  :  1135040
DOI  :  10.1186/1472-6750-12-37

 received in 2012-03-16, accepted in 2012-06-01,  发布年份 2012

【 摘 要 】

Background

The production of transgenic plants, either for the overproduction of the protein of interest, for promoter: reporter lines, or for the downregulation of genes is an important prerequisite in modern plant research but is also very time-consuming.

Results

We have produced additions to the pPZP family of vectors. Vector pPZP500 (derived from pPZP200) is devoid of NotI sites and vector pPZP600 (derived from pPZP500) contains a bacterial kanamycin resistance gene. Vector pMAA-Red contains a Pdf2.1: DsRed marker and a CaMV:: GUS cassette within the T-DNA and is useful for the production of promoter: GUS lines and overexpression lines. The Pdf2.1 promoter is expressed in seeds and syncytia induced by the beet cyst nematode Heterodera schachti in Arabidopsis roots. Transgenic seeds show red fluorescence which can be used for selection and the fluorescence level is indicative of the expression level of the transgene. The advantage is that plants can be grown on soil and that expression of the marker can be directly screened at the seed stage which saves time and resources. Due to the expression of the Pdf2.1: DsRed marker in syncytia, the vector is especially useful for the expression of a gene of interest in syncytia.

Conclusions

The vector pMAA-Red allows for fast and easy production of transgenic Arabidopsis plants with a strong expression level of the gene of interest.

【 授权许可】

   
2012 Ali et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Wang K (Ed): Agrobacterium Protocols. 2nd edition. Humana Press, Totowa, New Jersey; 2006.
• [2]Fraley RT, Rogers SG, Horsch RB: Genetic-Transformation in Higher-Plants. Crit Rev Plant Sci 1986, 4(1):1-46.
• [3]Bevan M: Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 1984, 12(22):8711-8721.
• [4]Lee LY, Gelvin SB: T-DNA binary vectors and systems. Plant Physiol 2008, 146(2):325-332.
• [5]Komori T, Imayama T, Kato N, Ishida Y, Ueki J, Komari T: Current status of binary vectors and superbinary vectors. Plant Physiol 2007, 145(4):1155-1160.
• [6]Hajdukiewicz P, Svab Z, Maliga P: The Small, Versatile Ppzp Family of Agrobacterium Binary Vectors for Plant Transformation. Plant Mol Biol 1994, 25(6):989-994.
• [7]pCambia Vectors. http://www.cambia.org/daisy/cambia/585 webcite
• [8]Szakasits D, Siddique S, Bohlmann H: An improved pPZP vector for Agrobacterium-mediated plant transformation. Plant Mol Biol Rep 2007, 25(3–4):115-120.
• [9]Yang W, Jefferson RA, Huttner E, Moore JM, Gagliano WB, Grossniklaus U: An egg apparatus-specific enhancer of Arabidopsis, identified by enhancer detection. Plant Physiol 2005, 139(3):1421-1432.
• [10]Yoo SY, Bomblies K, Yoo SK, Yang JW, Choi MS, Lee JS, Weigel D, Ahn JH: The 35 S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta 2005, 221(4):523-530.
• [11]Hadi MZ KE, Wendeler E, Reiss B: Simple and versatile selection of Arabidopsis transformants. Plant Cell Rep 135, 21:130.
• [12]Dhalluin K, Deblock M, Denecke J, Janssens J, Leemans J, Reynaerts A, Botterman J: The BAR gene as selectable and screenable marker in plant-engineering. Methods Enzymol 1992, 216:415-426.
• [13]Wenck A, Pugieux C, Turner M, Dunn M, Stacy C, Tiozzo A, Dunder E, van Grinsven E, Khan R, Sigareva M, et al.: Reef-coral proteins as visual, non-destructive reporters for plant transformation. Plant Cell Rep 2003, 22(4):244-251.
• [14]Baird GS, Zacharias DA, Tsien RY: Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 2000, 97(22):11984-11989.
• [15]Tzfira T, Tian GW, Lacroix B, Vyas S, Li JX, Leitner-Dagan Y, Krichevsky A, Taylor T, Vainstein A, Citovsky V: pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants. Plant Mol Biol 2005, 57(4):503-516.
• [16]Lin M-H, Gresshoff PM, Indrasumunar A, Ferguson BJ: pHairyRed: A Novel Binary Vector Containing the DsRed2 Reporter Gene for Visual Selection of Transgenic Hairy Roots. Molecular Plant 2011, 4(3):537-545.
• [17]Ckurshumova W, Caragea AE, Goldstein RS, Berleth T: Glow in the Dark: Fluorescent Proteins as Cell and Tissue-Specific Markers in Plants. Molecular Plant 2011, 4(5):794-804.
• [18]Goodin MM, Dietzgen RG, Schichnes D, Ruzin S, Jackson AO: pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J 2002, 31(3):375-383.
• [19]Matz MV: Fluorescent proteins from nonbioluminescent Anthozoa species (vol 17, pg 969, 1999). Nat Biotechnol 1999, 17(12):1227-1227.
• [20]Thomma BPHJ, Broekaert WF: Tissue-specific expression of plant defensin genes PDF2.1 and PDF2.2 in Arabidopsis thaliana. Plant Physiol Biochem 1998, 36(7):533-537.
• [21]Siddique S, Wieczorek K, Szakasits D, Kreil DP, Bohlmann H: The promoter of a plant defensin gene directs specific expression in nematode-induced syncytia in Arabidopsis roots. Plant Physiol and Biochem 2011, 49(10):1100-1107.
• [22]Jach G, Binot E, Frings S, Luxa K, Schell J: Use of red fluorescent protein from Discosoma sp (dsRED) as a reporter for plant gene expression. Plant J 2001, 28(4):483-491.
• [23]Szakasits D, Heinen P, Wieczorek K, Hofmann J, Wagner F, Kreil DP, Sykacek P, Grundler FMW, Bohlmann H: The transcriptome of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots. Plant J 2009, 57(5):771-784.
• [24]Holtorf S, Apel K, Bohlmann H: Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana. Plant Mol Biol 1995, 29(4):637-646.
• [25]Weisser P, Kramer R, Sprenger GA: Expression of the Escherichia coli pmi gene, encoding phosphomannose-isomerase in Zymomonas mobilis, leads to utilization of mannose as a novel growth substrate, which can be used as a selective marker. Appl Environ Microb 1996, 62:4155-4161.
• [26]Haldrup A, Noerremark M, Okkels F: Plant selection principle based on xylose isomerase. In Vitro Cellular & Developmental Biology-Plant 2001, 37(2):114-119.
• [27]Lu C, Fulda M, Wallis J, Browse J: A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. Plant J 2006, 45(5):847-856.
• [28]Butaye K, Goderis I, Wouters P, Pues J, Delaure S, Broekaert W, Depicker A, Cammue B, De Bolle M: Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions. Plant J 2004, 39(3):440-449.
• [29]Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY; 1989. 1, 2, 3
• [30]Holsters M, Dewaele D, Depicker A, Messens E, Vanmontagu M, Schell J: Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 1978, 163(2):181-187.
• [31]Voinnet O, Rivas S, Mestre P, Baulcombe D: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 2003, 33(5):949-956.
• [32]Logemann EBR, Ülker B, Somssich IE: An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods 2006., 2(16)
• [33]Jefferson RA, Kavanagh TA, Bevan MW: Gus Fusions - Beta-Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher-Plants. EMBO J 1987, 6(13):3901-3907.
• [34]Sijmons PC, Grundler FMW, Vonmende N, Burrows PR, Wyss U: Arabidopsis-Thaliana as a New Model Host for Plant-Parasitic Nematodes. Plant J 1991, 1(2):245-254.