Vectors, microorganisms and cDNA libraries

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2.1.7 Vectors, microorganisms and cDNA libraries

31 constructed by Dr. G. Jach (Max-Planck-Institute, Cologne, Germany) and was used for protein localization analysis (Willige et al. 2009). Since it also contains a dual CaMV35S promoter and a translational enhancer, the vector was also used to make overexpression constructs in this study.

2.1.7.1.5 pET28a

This vector carries an N-terminal His-Tag/thrombin/T7-Tag configuration plus an optional C-terminal His-Tag sequence and a Kan^R gene that codes for kanamycin resistance. It was used for His-tagged protein overexpression (Novagen, Darmstadt, Germany).

2.1.7.1.6 pRS300

This vector is a backbone for expressing plant artificial miRNAs, it contains the miR319a precursor which was cloned via the SmaI site in pBSK and an Amp^R gene that confers ampicillin resistance. This vector was obtained from Dr. Detlef Weigel (Max Planck Institute for Developmental Biology, Tübingen, Germany).

2.1.7.1.7 R4L1pDEST_LacZi (Mitsuda et al. 2010)

This plasmid is a derivative of the yeast integration and reporter vector pLacZi (Clontech/TAKARA) into which an unconventional gateway cassette attR4-ccdB/Cm^r-attL1 has been introduced for cloning (Mitsuda et al. 2010). The cassette was inserted into the SmaI site in front of the reporter gene LacZ under the control of the iso-1-cytochrome c promoter from yeast.

The lethal ccdB gene serves for positive selection during cloning with BP and LR clonase. A bla gene for ampicillin resistance and a Col E1 ori allows the propagation of the vector in E.

coli. URA3 is used as a selectable marker for the integration of the linearized vector into the yeast genome.

2.1.7.1.8 R4L1pDEST_HISi (Mitsuda et al. 2010)

This plasmid is a derivative of the yeast integration and reporter vector pHISi (Clontech/TAKARA) into which an unconventional gateway cassette attR4-ccdB/Cm^r-attL1

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has been introduced for cloning (Mitsuda et al. 2010). The cassette was inserted into the SmaI-SacII site in front of the reporter gene HIS3 under the control of a minimal promoter of HIS3.

The lethal ccdB gene serves for positive selection during cloning with BP and LR clonase. A bla gene for ampicillin resistance and a Col E1 ori allows the propagation of the vector in E.

coli. HIS3 or URA3 are used as selectable markers for the integration of the linearized vector into the yeast genome.

2.1.7.1.9 pDEST_GAD424 (Mitsuda et al. 2010)

This plasmid is a derivative of the vector pGAD424 (Clontech/TAKARA) into which a conventional gateway cassette attR1-ccdB/Cm^r-attR2, followed by three in-frame stop codons was inserted via the SmaI site (Mitsuda et al. 2010). This plasmid is the carrier of a library of 1,498 transcription factors, which was used for the yeast one-hybrid screening. cDNA of selected transcription factors without a stop codon was introduced through a Gateway system into the vector. Each TF gene was thereby fused to the activation domain of the yeast GAL4 transcription activator. It contains pMB1 ori and ampicillin resistance (bla) for propagation in E. coli. Gene LEU2 serves for selection in yeast. A minimum ADH1 promoter provides a low expression of the fusion protein and the SV40 T-anti gene nuclear localization sequence is used for targeting the protein into the yeast nucleus (Chien et al. 1991).

2.1.7.1.10 pAS2-1 (Harper et al. 1993)

This plasmid was used to construct the yeast two-hybrid protein-bait. It generates a fusion of the GAL4DNA-BD and a protein of interest that cloned into the MCS in the correct reading frame. pAS2-1 carries the wild-type yeast CYHS2 gene, which confers sensitivity to cycloheximide in transformed cells. pAS2-1 is a shuttle vector that replicates autonomously in both E.coli and S. cerevisiae and carries the bla gene, which confers ampicillin resistance in E.coli. It also contains the TRP1 nutritional gene that allows yeast auxotrophs to grow on limiting synthetic media.

2.1.7.1.11 pACT2 (Li et al. 1994)

This plasmid is used for establishing library for yeast two-hybrid screening. It generates a fusion of the GAL4-AD, a HA epitope tag, and a protein of interest (or protein encoded by a

33 cDNA in a fusion library) cloned into the MCS in the correct reading frame. pACT2, which is derived from pACT (1), contains a unique EcoR I site in the MCS. The hybrid protein is expressed at high levels in yeast host cells from the constitutive ADH1 promoter; transcription is terminated at the ADH1 transcription termination signal. The protein is targeted to the yeast nucleus by the nuclear localization sequence from the SV40 T-antigen which has been cloned into the 5' end of the GAL4 AD sequence. pACT2 is a shuttle vector that replicates autonomously in both E. coli and S. cerevisiae and carries the bla gene which confers ampicillin resistance in E. coli. pACT2 also contains the LEU2 nutritional gene that allows yeast auxotrophs to grow on limiting synthetic media.

2.1.7.2 Microorganisms

2.1.7.2.1 Escherichia coli DH10B (Lorow and Jessee 1990)

Genotype: F^– endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ^–.

This strain was used as host strain for cloning.

2.1.7.2.2 Escherichia coli BL21 (Pharmacia, Freiburg)

Genotype: F^– ompT gal dcm lon hsdSB(rB– mB–) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]).

This strain was used to express recombinant proteins.

2.1.7.2.3 Escherichia coli DB3.1 (Invitrogen)

Genotype: F^– gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(rB–, mB–) ara14 galK2 lacY1 proA2 rpsL20(Sm^r) xyl5 Δleu mtl1.

E. coli DB3.1 contains a specific mutation in the DNA gyrase (gyrA462) that makes it resistant to the lethal ccdB gene product. It was used as a host strain for propagation of the two plasmids R4L1pDEST_lacZi and R4L1pDEST_HISi.

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2.1.7.2.4 Agrobacterium tumefaciens GV3101/pmP90RK (Koncz and Schell 1986) Genotype: C58C1 pMK90RK, Rifr, Gmr, Kmr.

This strain was used for transformation of wild-type A. thaliana (ecotype Col-0) and wild-type E. salsugineum (ecotype Shandong) plants.

2.1.7.2.5 Saccharomyces cerevisiae YM4271 (Liu et al. 1993)

Genotype: MATa, ura3-52, his3-200, ade2-101, lys2-801, leu2-3, 112, trp1-901, Tyr1-501, gal4D, gal8D, ade5 :: hisG.

This yeast strain has been used for yeast one-hybrid screening. It contains mutations in different genes, such as ura3, his3, and leu2. Because of these mutations, the strain loses the ability to synthesize the corresponding enzymes or amino acids. Thus, uracil, leucine and histidine can be used as selection markers.

2.1.7.2.6 Saccharomyces cerevisiae Y190 (Durfee et al. 1993)

Genotype: MATa, gal4-542, gal80-538, his3-200, trp1-901, ade2-101, ura3-52, leu2-3,112, URA3:: GAL-LacZ, Lys2::GAL1-HIS3,cyh^r)

This yeast strain has been used for yeast two-hybrid screening. It contains mutations in different genes, such as try3, his3, and leu2. Because of these mutations, the strain loses the ability to synthesize the corresponding enzymes or amino acids. Thus, tryptophan, leucine, and histidine can be used as selection markers.

2.1.7.3 cDNA libraries

The cDNA library that was used for yeast two-hybrid analysis was kindly provided by Dr.

Csaba Koncz (Max plank institute for Plant Breeding Research, Cologne, Germany). This library was an oligo (dT) primed cDNA library prepared in the plasmid pACT2 using mRNA from an A. thaliana cell suspension. The other cDNA library that was used for yeast one-hybrid analysis was obtained from Dr. Nobutaka Mitsuda (National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan). This library is composed only of 1,498 TFs from A. thaliana and is cloned in the plasmid pDEST_GAD424 by gateway cloning.

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