DNA‐Vectors

dried seeds were collected and sowed on soil. To detect double-knockout plants, young plants were analysed by PCR-screening on gDNA-level.

2.1.2.10 Harvesting of A. thaliana seeds

Seeds from A. thaliana were harvested after drying and opening of the siliques. The seeds were wiped off with clean fingers over clean paper and sieved to remove large particles. For fine purification, seeds were transferred to another sheet of paper so that small flower particles could glue onto the paper. Residual material was carefully blown away. The seeds were transferred into a 1.5 mL Eppendorf tube, covered with parafilm with tiny holes and dried over night. Storage of seeds was performed in closed 1.5 mL Eppendorf tubes at 4 °C.

2.5 Molecular biologic methods

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proteins were expressed under control of the strong cauliflower mosaic virus 35S promoter (CaMV35S). The vector p21GT contains the short CTS (cytoplasmic – transmembrane – stem) region of a glycosyltransferase (GT) which was excised using XbaI and SacI or SpeI. Both vectors enabled production of fusion proteins for further purification of expressed proteins by affinity chromatography and/or detection by western blot. Vector p21GT contains a C-terminal His-tag whereas ppT8 contains a C- terminal myc-tag (see Figure 12). Both vectors contain kanamycin resistance genes in the vector backbone as well as in the T-DNA region (which is therein driven by the nopaline synthase promoter) to allow selection of bacteria during cloning as well as screening of genetically modified Arabidopsis plants on plant medium supplemented with 50 µg/mL kanamycin (Table 3).

For Subcellular localisation studies by confocal laser scanning microscopy (CLSM), the investigated gene was expressed in the plant binary expression vector p20F. This vector is very similar to p21 but contains a C-terminal GFP-tag. p20F is also derived from pPT2, as described by Strasser et al., 2007. Due to weak expression, the vector had to be modified during this work. In order to gain an increased expression, the 35S-promoter was replaced by an enhanced version to give vector p20F eCaMV3S.

This modified enhanced cauliflower mosaic virus 35S-promoter (eCaMV35S) contains a tobacco mosaic virus–Ω translation enhancer, a 5’-UTR (Batoko et al., 2000; Gallie, 2002). The encoding promoter sequence was cloned from vector p30GALT1_Fc-GFP which was kindly supplied by Dr. Richard Strasser, DAGZ, BOKU and is basically derived from vector pVKH18En6 (Saint-Jore et al., 2002).

Figure 12: Overview about plant binary expression vectors used during this work.  

The T‐DNA region present in all three vectors is flanked by left (LB) and right border (RB) and contains CaMV35S  promoter, g7T terminators together with a multiple cloning site  (MCS), and a  kanamycin resistance gene  (neomycin phosphotransferase, npt). A) Vector p21GT; His‐tag adjacent to the MCS; B) Vector ppT8; Myc‐tag  adjacent to the MCS; C) Vector p20F eCaMV35S; GFP adjacent to the MCS. The common 35S promoter has been  replaced by an enhanced 35S promoter (eCaMV35S) together with a viral enhancer element (5’UTR from TMV,  called Ω‐sequence). Source of sequence: Dr. Richard Strasser, DAGZ, BOKU, modified. 

2.1.3.3 Vectors for down regulation of genes by RNAi silencing

Vectors used for down regulation of the GME gene were pSAT- and plant binary expression vector pRCS2-ocs-nptII which were described in detail (Tzfira et al., 2005;

Dafny-Yelin et al., 2007). This system includes two types of vectors: pSAT-vectors used for creation of the RNAi expression cassette, which is then transferred into the T-DNA region of the expression vector pRCS2. Table 6: gives an overview about both vectors used and their features. Figure 13 illustrates the structure of pSAT4-35SP.RNAi used for cloning of GME RNAi as well as pRCS2-ocs-nptII vector used for expression in plants. As illustrated in Figure 13 A, the expression cassette contains two multiple cloning sites and a ChsA-intron in between, two CaMV 35S promoter, a translational enhancer element, antibiotic resistance gene and terminator. In order to transfer the

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cassette into the expression vector, the cassette is flanked by two recognition sites of the rare cutter I-SceI, a homing endonuclease. The corresponding recognition sites of I- SceI and other rare cutters are also present in the multiple cloning site of pRCS2-ocs- nptII. The T-DNA region of pRCS2-ocs-nptII contains a multiple cloning site and OCS activator, promoter and terminator for the kanamycin resistance gene nptII (see Table 6and Figure 13 B).

Promoter &

enhancer

antibiotic

resistance gene terminator rare cutters

pSAT4-35SP.RNAi

2x 35S CaMV; 5' UTR from tobacco

etch virus (translational

enhancer)

ampicillin resistance gene

CaMV 35S terminator

cassette flanking:

I –SceI

pRCS2-ocs-nptII OCS activator;

OCS promoter

kanamycin

resistance gene OCS terminator

rare cutters present in the MCS:

I-PpoI I-SceI I-CeuI PI-PspI

Table  6:  Overview  about  main  features  of  both  pSAT4‐35SP.RNAi  and  pRCS2‐ocs‐nptII  vectors.  

Source: Tzfira et al. (2005); Dafny‐Yelin et al. (2007); NCBI. 

  Figure 13: Vectors used for silencing of GME by RNAi technique.  

A) Expression cassette of a pSAT‐vector, composed of 2x CaMV35S promoters, a translational enhancer (5’UTR  from tobacco etch virus), two multiple cloning sites (MCS1 and II) in which the RNAi sequence has to be cloned, a  ChsA‐intron between the two MCS, and a CaMV 35S terminator. B) Cloning of the expression cassette into the T‐

DNA region of the binary expression vector pRCS2‐ocs‐nptII. The T‐DNA region is flanked by left (LB) and right  border (RB) and contains the MCS, OCS activator, OCS promoter and activator together with kanamycin resistance  gene (nptII) and OCS terminator. Source of figure: Tzfira et al. (2005); Dafny‐Yelin et al. (2007); Sequence from  NCBI; modified.