Expression analysis by Northern Blot: - Expression analysis of selected genes in wheat cultivar

3. RESULTS:

3.2 Identification of Genes for Disease Inducible Promoter identification from wheat:

3.2.1 Expression analysis of selected genes in wheat cultivar ―Florida‖:

3.2.1.2 Expression analysis by Northern Blot:

On the basis of the results taken through PCR the four selected genes were checked for the up regulation of expression upon F. graminearum inoculation. Small fragments were amplified from all the four genes and cloned in Topo-Ta cloning vector. The fragments were separated from the plasmids and eluted out of the agarose gel. These fragments were then radio actively labelled with P³² and used in the Northern Blot analysis for all the genes under study. The process of Northern Blot analysis is stated in section 2.2.5.6. A total of four RNA gels were prepared for RNA harvested from respective treatments. RNA was taken from the same test tube for a treatment on all the four gels.

Figure: 3.45a Northern Blot analysis for pk0023 gene.

M E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 pk0023

Figure: 3.45b control gel for Northern Blot analysis of pk0023

Freshly isolated RNA from mock inoculated and inoculated probes were run on 1% degeneration agarose gell and RNA was transferred to the nylon membrane by capillary method and cross linked. Pk0023 specific 400 bp long single stranded radio actively labelled DNA probe was later used to hybridize RNA specific to pk0023 gene. The results were seen on the X-rays film.

M= High range RNA marker, C= Control plant with out inoculation, 1-7 mock inoculated probes harvested 12hai, 24hai, 48hai..and 144hai. 8-14 F. graminearum inoculated probes.

M E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Mock inoculations Inoculations

pk-0023

3 kb 2 kb

The results showed the same trend as it was shown in the last section where the expression was studied by PCR. On three out of four selected genes there was no indication of any expression for mock controls. On F. graminearum inoculated probes the expression seems to start atleast 48 hai and the maximum expression is found at 72 hai and 96 hai which keeps on decreasing till 144 hai.

In terms of the quantity of the transcript pK0023 seemed to express abundantly. Slight expression started at 48 hai and increased abruptly at 72hai and then started decreasing after that.

Figure:3.46a Northern Blot analysis for PDR like ABC Transporter gene

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PDR Like ABC Transporter Gene

Figure 3.56b control Gel for4 Northern Blot analysis of PDR like ABC Transporter gene

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Mock Inoculations Inoculations PDR like ABC Transporter gene

3 kb 2 kb

300b 200b

long single stranded radio actively labelled DNA probe was later used to hybridize RNA specific to ―PDR Like ABC Transporter Gene‖. The results were seen on the X-rays film.

M= High range RNA marker, C= Control plant with out inoculation, 1-7 mock inoculated probes harvested 12hai, 24hai, 48hai .and 144hai. 8-14 F. graminearum inoculated probes.

The expression of PDR like ABC transporter gene was also differential for inoculated and mock inoculated control but the expression level was seemingly low as compared to pk0023.

At the same time there seemed very little spots in the mock control lanes on the film which indicated little expression at mock control as well.

The expression of ―Oxalate oxidase precursor gene‖ was absolutely differential and there were no signs of expression on non inoculated control as well as on the mock control.

Expression started at 72 hai and went upto 144 hai but the level of expression was very low compared to other genes.

In case of ―chitinase gene‖ the story was a little bit different; here we can see the differential expression of the gene, expression gets very high at 72 hai and 96 hai and then decreases a bit going to 144 hai with a little change, the expression level is high as well. At the same time we can also see some expression in all the lanes with mock control although this expression is very low as compared to inoculated ones.

Figure: 3.47a Northern Blot analysis for Oxalate Oxidase precursor gene.

M E 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Mock Inoculations Inoculations Oxalate oxidase precursor

300b 200b

M E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Oxalate Oxidase precursor

Figure: 3.47b control gel for Northern Blot analysis for Oxalate Oxidase precursor gene.

Freshly isolated RNA from mock inoculated and inoculated probes were run on 1% degeneration agarose gell and RNA was transferred to the nylon membrane by capillary method and cross linked. ―Oxalate oxidase precursor‖ specific 400 bp long single stranded radioactively labelled DNA probe was later used to hybridize RNA specific to ―Oxalate oxidase precursor ‖.

The results were seen on the X-rays film.

M= High range RNA marker, C= Control plant with out inoculation, 1-7 mock inoculated probes harvested 12hai, 24hai, 48hai .and 144hai. 8-14 F. graminearum inoculated probes.

Figure: 3.48a Northern Blot analysis for chitinase gene.

M E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Mock Inoculations Inoculations

Ta-Chitinase

3 kb 2 kb

300b 200b

M E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Ta-Chitinase

Figure: 3.48b Control gel for Northern Blot analysis of Ta-chitinase gene.

Freshly isolated RNA from mock inoculated and inoculated probes were run on 1% degeneration agarose gell and RNA was transferred to the nylon membrane by capillary method and cross linked. ―Ta-Chitinase‖ specific 400 bp long single stranded radio actively labelled DNA probe was later used to hybridize RNA specific to ―Ta-Chitinase‖. The results were seen on the X-rays film.

M= High range RNA marker, C= Control plant with out inoculation, 1-7 mock inoculated probes harvested 12hai, 24hai, 48hai .and 144hai. 8-14 F. graminearum inoculated probes.

All the four genes studied above seem suitable candidates for finding promoters as the promoters which are regulating their expression are sensitive for F. graminearum infection.

―pk0023‖ is a best candidate as it not only expresses differentially but the expression level is very high as well. The differential expression level of ―Chitinase‖ as well as of ―PDR like ABC transporter gene‖ is high as well but there seems some expression at mock inoculations which may weaken their case. ―Oxalate Oxidase Precursor gene‖ is although differentially expressed and there is no expression on mock inoculation but the expression level is too low.

3 kb 2 kb

Table: 3.6. Performance of over expression lines to FHB and PM

Transgenic Line Change in resistance to FHB Change in resistance to PM Over expression lines (under

the Ubi-1 promoter)

I.A-1 Not evaluated ++

I.A-2 Not evaluated Not evaluated

I.A-3 ++ +++

I.A-4 Not evaluated ++

I.A-5 Not evaluated Not evaluated

I.A-6 + ++

I.A-17 Not evaluated Not evaluated

I.A-18 + +++

Over expression lines (under the Vst-1 promoter)

I.A-7 Not evaluated +

I.A-8 ++ -

I.A-9 Not evaluated --

I.A-10 Not evaluated -

I.A-11 + ++++

I.A-12 ++ ++

Note: ++++= more 69% increase in resistance, +++= 50-69% increase in resistance, ++= 30-49% increase in resistance, +=

10-29% increase in resistance, -= 10% decrease to 10% increase in resistance, --= more than 10% decrease in resistance.

Im Dokument Reverse and Forward Genetic Approaches for the Development of Disease Resistant Wheat (Triticum aestivum L.) (Seite 105-112)