Obtaining of knockout plants

Both knockout-lines for AtFUC95A and AtFXG1 were obtained from NASC as segregating T3 plants having one T-DNA insertion in an exon. The first part of the work was to obtain knockout homozygous lines. In a second time the confirmation of the knockout status of these plants at the genomic and cDNA level has been undertaken.

3.2.1.1 Identification of AtFUC95A and AtFXG1 knockout plants

Both knockout-lines of AtFUC95A (GK-440B01-018208) and AtFXG1 (SALK_016739) were describes as segregating T3 plants having one T-DNA insertion in

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an exon. Therefore a direct screening for homozygous knockout plants has been performed using gene specific primers flanking the T-DNA insertion point. T-DNA flanking sequence information from www.Arabidopsis.org was used for estimation of insertion point and for primer design. Figure 17 gives an overview about the gDNA- structure including primer annealing site of AtFUC95A and AtFXG1 based on TAIR.

Figure 17: Genomic DNA sequence of A. thaliana AtFXG1 (At1g67830) and AtFUC95A (At4g34260).  

Both genes are composed of 5’ and 3’ UTR’s (gray) and several exons (green) and introns (lines). AtFXG1 is  composed of three exons and two introns and AtFUC95A of 9 exons and 8 introns. The position of ATG and stop  codon is indicated as well as the position of T‐DNA insertion flanking sequence (TAIR, NASC). Magenta arrows are  primers used for screening of knockout plants. After sequencing the insertion point has been evaluated and its  position is shown in base pairs downstream of the ATG. 

Genomic DNA was isolated from young leaves of putative knockout plants for AtFXG1 and AtFUC95A. The isolated gDNA was then used to screen the plants for the presence of a T-DNA insertion inside the genes of interest with two gene specific, T- DNA insertion flanking primers (Figure 18 A and B). A signal of 884 bp was expected for AtFXG1 in wildtype (Figure 18 A, lane 3) and heterozygous plants and a signal of 1.353 bp for AtFUC95A (Figure 18 B, lane 3). An absence of signal was expected in the case of homozygous knockout plants. For identification of knockouts for AtFUC95A (At4g342609), twenty one plants of line GK-440B01 were screened and five plants were identified as putative knockouts of which plant #13 (designated Atfuc95A#13) was further studied. Twenty nine plants of AtFXG1 (At1g67830) knockout line SALK_016739 were screened and one plant, designated as Atfxg1#27, was identified as a putative knockout. To obtain confirmation of the identification of the knock-out plants, a PCR was performed on the selected putative knockout plants with one gene specific primer and the T-DNA specific primer LBa1 which anneals to the left border. This screening has

been repeated on one plant in the offspring generation (AtFUC95A knockout plant 3, designated Atfuc95A#13/3, and AtFXG1 knockout plant 1, designated Atfxg1 #27/1).

Screening of AtFXG1 knockout plants was performed with the flanking primer AtFXG1ovA3 and the left border specific primer LBa1 (Figure 18 C, lane 2) and in case of AtFuc95A with the flanking primers 440B01A5 and LB-GABI-KAT_new (Figure 18 C, lane 3). A signal was then detected in the knockout plants. This gave a clear confirmation of presence of a T-DNA insertion. As a last step, the resulting fragments were purified and sequenced.

Figure 18: Identification of homozygous T‐DNA insertion lines of the Atfxg1 knockout and Atfuc95A knockout in  the offspring generation.  

A) Screening for identification of homozygous knockouts of Atfxg1using T‐DNA flanking primers, giving a signal of  884 bp in the wildtype control (lane 3). No signal was observed in the knockout plant Atfxg1#27/1 (lane 2). B)  Screening for identification of homozygous knockouts of the Atfuc95A knockout using T‐DNA flanking primers,  giving a signal of 1.353 bp in the wildtype control (lane 3). No signal was observed in the knockout plant  Atfuc95A#13/3 (lane 2). C) Confirmation of presence of T‐DNA left border using one flanking‐ and a left border  specific primer. Signals were obtained in Atfxg1#27/1 and Atfuc95A#13/3 which confirm presence of T‐DNA left  border. Lane 1 – Fermentas 100 bp DNA Ladder Plus. Col‐0 WT – Columbia wildtype was used as a control. 

3.2.1.2 Confirmation of T-DNA insertion by sequencing

A fragment of gDNA and T-DNA left border of Atfuc95A knockout was amplified with primers 440B01A5 and LB-GABI-Kat_new and sequenced (Figure 17 B and Figure 19, lane 3). The gene fragment of the Atfxg1 knockout was amplified using primers AtFXG1ovA3 and LBa1 and then sequenced (see Figure 17 A and Figure 19, lane 2).

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Figure 19: DNA fragments of all identified T‐DNA insertion lines amplified with gene specific primers and left  border primer.  

Lane 2: Atfxg1#27/1, primers AtFXG1ovA3 and LBa1; lane 3: Atfuc95A#13/3, primers 440B01A5 and LB‐GABI‐

Kat_new. Lane 1 – Fermentas 100 bp DNA Ladder Plus. 

Analysis of the sequencing result confirmed the presence and identity of a T-DNA left border. For Atfuc95A, the insertion point has been found approximately 2.142 bp downstream the initiating codon (Figure 17 B) in exon 5. In the case of Atfxg1 the insertion point has been identified about 1.222 bp downstream the initiating codon in exon 5 close to the end (Figure 17 A).

3.2.1.3 Confirmation of knockout status at the cDNA level

To confirm the absence of any gene product in the knockout plants, RNA was isolated from leaves of Atfuc95A#13/3, Atfxg1#27/1 as well as Col-0 wildtype (positive control) and converted into cDNA by reverse-transcription. PCR screening followed on Atfuc95A using primers Fu2ovA5 and Fuc2ovA3 which gave a signal in wildtype control of 2.550 bp (Figure 20 A, lane 3).

Atfxg1 was screened using primers AtFXG1ovA5 and AtFXG1ovA3, giving a signal in wildtype control of 1.132 bp (Figure 20 B, lane 3).

No cDNA could be detected in both knockout lines whereas a signal of expected size was achieved in Col-0 wildtype plants, thus confirming the knockout status of the selected plants.

  Figure 20: Confirmation of absence of gene product in knockout lines on cDNA level by PCR.  

A) Knockout line Atfuc95A#13/3 (lane 2) and CTRL Col‐0 WT (lane 3) amplified with cloning primers (2.550 bp in  the wildtype control). B) AtFXG1 knockout line Atfxg1#27/1 (lane 2) and CTRL Col‐0 WT (lane 3) amplified with  cloning primers (1.132 bp in the wildtype control). Template cDNA was from leaves. Lane 1 – Fermentas 100 bp  DNA Ladder Plus. CTRL Col‐0 WT – Control Col‐0 wildtype. 

3.2.2 Xyloglucan profile of AtFUC95A knockout plants but not of AtFXG1