cDNA-AFLP ANALYSIS OF GENE EXPRESSION IN RED RASPBERRY
(Rubus idaeus L.) DURING WATER STRESS
ANALIZA EXPRESIEI GENICE LA ZMEUR (Rubus idaeus L.)
ÎN CONDIŢII DE STRES HIDRIC PRIN TEHNICA cDNA-AFLP
CIOBOTARI Gh.1, EFROSE Rodica Catalina1, BRINZA Maria1, SFICHI-DUKE Liliana1
1University
of Agricultural Sciences and Veterinary Medicine of Iasi, Romania
INTRODUCTION
According to the World Atlas of Desertification, UNEP (United Nations Environment
Programme), dry lands cover 40% of the world’s land surface (almost 5.1 billion ha), and they
are the habitat and source of livelihood for about 1 billion people. With the growth of
population and the development of modern agro-industry and the other industries, extension
of the dry land surfaces becomes a growing concern all over the world. This concern is
reflected in the Convention on Biological Diversity’s dry lands work program, and in the
establishment of the UN Convention to Combat Desertification. To mitigate the effects of
drought, it is very important first to promote the effective and efficient use of existing plant
resources, second to investigate the physiological processes and mechanisms that help plants
to acclimatize to the water deficiency conditions. It is assumed that molecules and compounds
such as proline, glycine betaine and soluble sugars etc. synthesized and accumulated during
desiccation play an important role in the protection of the plants from stress . However, at this
time the molecular basis for these protective mechanisms is almost unknown. In this
experiment, the screening of the differentially expressed genes in water deficiency growth
conditions was accomplished by means of the high reproductive technique (cDNA-AFLP). By
this method we identified in stressed plants multiple polymorphic transcript-derived fragments
(TDFs) that highlight specific plant reactions on molecular level.
MATERIAL AND METHODS
Plant material
Raspberry (Rubus idaeus L.) plants were grown in the greenhouse of the “V. Adavachi” Research
Station (University of Agricultural Sciences and Veterinary Medicine of Iasi). Stressed plants were
grown on the soil with 35% water hydration, and control plants were normal hydrated. Raspberry
leaves were collected from mature greenhouse-grown plants and were immediately frozen in liquid
nitrogen and stored at −80 oC.
RNA extraction
Total RNA was isolated from raspberry very fine grinded leaves, in liquid nitrogen, with the
SpectrumPlantTotal RNA Kit according to the manufacturer’s protocol. RNA quality was checked
using Bioanalyzer 2100 and RNA 6000 Nano Kit that allow visualization of 18S and 28S subunits
and calculation of RNA Integrity Number (RIN)
First-strand cDNA synthesis
The first-strand cDNA synthesis was accomplished with SuperScript II Reverse Transcriptase (RT)
Kit that is an engineered version of MMLV RT with reduced RNase H activity and increased thermal
stability.
Second-strand cDNA synthesis
The second-strand cDNA synthesis was performed with the SuperScript Double-Stranded cDNA
Synthesis Kit that contains all of the reagents, except an oligo(dT) containing primer, necessary to
make double-stranded cDNA from total RNA or poly A+ selected RNA (mRNA).
The next steps of cDNA-AFLP analysis were made with AFLP Analysis System I, AFLP Starter
Primer Kit according to the manufacturer’s protocols.
Restriction Endonuclease Digestion
To prepare an AFLP template, cDNA is digested with two restriction endonucleases
simultaneously. This step generates the required substrate for ligation and subsequent
amplification. The restriction fragments for amplification are generated by two restriction
endonucleases: EcoR I and Mse I. EcoR I has a 6-bp recognition site, and Mse I has a 4-bp
recognition site. When used together, these enzymes generate small DNA fragments that will
amplify well and are in the optimal size range (<1 kb) for separation on denaturing
polyacrylamide gels. Due to primer design and amplification strategy, these EcoR I – Mse I
fragments are preferentially amplified (rather than EcoR I – EcoR I or Mse I – Mse I fragments).
Ligation of Adapters
After heat inactivation of the restriction endonucleases, the cDNA fragments are ligated to EcoR I
and Mse I adapters to generate template DNA for amplification. These common adapter
sequences flanking variable cDNA sequences serve as primer binding sites on these restriction
fragments. Using this strategy, it is possible to amplify many DNA fragments without having prior
sequence knowledge.
Amplification Reactions
PCR was performed in two consecutive steps. In the first step, called preamplification, cDNAs are amplified with AFLP
primers each having one selective nucleotide. The PCR products of the preamplification reaction was diluted and
used as a template for the selective amplification using two AFLP primers (the EcoR I selective primer and Mse I
selective primer), each containing three selective nucleotides. This two-step amplification strategy generated enough
templates DNA for thousands of AFLP reactions. The selective primers in the AFLP Analysis System I contain three
selective nucleotides. In practice, using the AFLP Analysis System I with plants having genomes ranging in size from 5
× 108 to 6 × 109 bp, the number of fragments amplified per sample per primer pair averages 50, but may range from
as low as 10 to ~ 100 depending on the sequence context of the selective nucleotides, and the complexity of the
genome.
PCR preamplification reactions program performed 20 cycles at:
94oC for 30 s
56oC for 60 s
72oC for 60 s
PCR selective amplification reactions program performed 33 cycles at:
A. One cycle at 94°C for 30 s; 65°C for 30 s; and 72°C for 60 s;
B. Lowered the annealing temperature each cycle 1°C during 9 cycles. This gives a touchdown phase of 10 cycles;
C. The last 23 cycles at:
94°C for 30 s;
56°C for 30 s;
72°C for 60 s.
Separation of Amplified Fragments on Denaturing Polyacrylamide Gels
Products from the selective amplification was separated on a 4% agarose gel. The resultant banding
pattern was manually analyzed for polymorphic transcript-derived fragments (TDFs).
RESULTS
0 1
2
3 4 5
6
7 8
9 10 11 12 13 14
0 15 16 17 18 19 20 21 22 23 24 25 26 27 28
06.10.2011/Amplificare Selectiva probele 109 si 115
35% hidratare (109-stres, 115 -martor) – Opal - sol/turba
0
Ladder
1
109
2
115
3
109
4
115
5
109
6
115
7
109
8
115
9
109
10
115
11
109
12
115
13
109
14
115
0
Ladder
15
109
16
115
17
109
18
115
19
109
20
115
21
109
22
115
23
109
24
115
25
109
26
115
27
109
28
115
E-ACC / M-CAG
E-ACC / M-CAC
E-ACC / M-CAT
E-ACC / M-CTT
E-ACC / M-CTG
E-ACT / M-CAA
E-ACT / M-CAC
E-ACT / M-CTA
E-ACT / M-CAT
E-ACT / M-CTC
E-ACT / M-CTG
E-ACT / M-CAG
E-AAG / M-CAG
E-AAG / M-CAA
0 29 30 31 32 33 34 35 36 37 38 39 40 41 42
0 43 44 45 46 47 48 49 50
06.10.2011/Amplificare Selectiva probele 109 si 115
35% hidratare (109-stres, 115 -martor) – Opal - sol/turba
0
Ladder
29
109
30
115
31
109
32
115
33
109
34
115
35
109
36
115
37
109
38
115
39
109
40
115
41
109
42
115
0
Ladder
43
109
44
115
45
109
46
115
47
109
48
115
49
109
50
115
E-AAG / M-CAC
E-AAG / M-CAT
E-AAG / M-CTC
E-AAG / M-CTT
E-AGG / M-CAT
E-AGG / M-CAA
E-AGG / M-CTA
E-AGG / M-CTC
E-AGG / M-CTT
E-ACA / M-CTA
E-ACA / M-CAT
0
1
2
3 4
5
6
7
8
9 10 11 12 13 14
0 15 16 17 18 19 20 21 22 23 24 25 26 27 28
07.10.2011/Amplificare Selectiva probele 136 si 137
35% hidratare (136-stres, 137-martor) – Cayuga - sol/turba
0
Ladder
1
136
2
137
3
136
4
137
5
136
6
137
7
136
8
137
9
136
10
137
11
136
12
137
13
136
14
137
0
Ladder
15
136
16
137
17
136
18
137
19
136
20
137
21
136
22
137
23
136
24
137
25
136
26
137
27
136
28
137
E-ACC / M-CAG
E-ACC / M-CAC
E-ACC / M-CAT
E-ACC / M-CTT
E-ACC / M-CTG
E-ACT / M-CAA
E-ACT / M-CAC
E-ACT / M-CTA
E-ACT / M-CAT
E-ACT / M-CTC
E-ACT / M-CTG
E-ACT / M-CAG
E-AAG / M-CAG
E-AAG / M-CAA
0 29 30 31 32 33 34 35 36 37 38 39 40 41 42
07.10.2011/Amplificare Selectiva probele 136 si 137
35% hidratare (136-martor, 137-stres) – Cayuga - sol/turba
0 43 44 45 46 47 48 49 50
0
Ladder
29
136
30
137
31
136
32
137
33
136
34
137
35
136
36
137
37
136
38
137
39
136
40
137
41
136
42
137
0
Ladder
43
136
44
137
45
136
46
137
47
136
48
137
49
136
50
137
E-AAG / M-CAC
E-AAG / M-CAT
E-AAG / M-CTC
E-AAG / M-CTT
E-AGG / M-CAT
E-AGG / M-CAA
E-AGG / M-CTA
E-AGG / M-CTC
E-AGG / M-CTT
E-ACA / M-CTA
E-ACA / M-CAT
CONCLUSIONS
Screening 64 primer combinations identified multiple transcript-derived fragments
(TDFs) that are differentially expressed in water stress conditions. The differences
between control and stressed plants were qualitative when TDFs were either
present or absent or quantitative when TDFs showed different levels of expression.
The results show that cDNA-AFLP is a valuable technique for studying expression
patterns of genes involved in sensitivity/tolerance mechanisms to water stress in red
raspberry
Acknowledgments
This study has been financed by the National Authority for Scientific Research,
Operational Program POSCCE, ID 524, contract no. 151/2010, project title “The
significance of the relationship among genomic response, phenylpropanoid
metabolism and photosynthesis for the optimization of biosynthetic potential of
raspberry and blackberry cultivars in abiotic stress conditions”
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