ГЕНЕТИКА, 2012, том 48, № 2, с. 204-210
ГЕНЕТИКА РАСТЕНИЙ
УДК 575.1:582.783
SUPPRESSIVE SUBTRACTIVE HYBRIDIZATION METHOD ANALYSIS AND ITS APPLICATION TO SALT STRESS IN GRAPEVINE (Vitis vinifera L.)
© 2012 г. S. Daldoul1,2, A. Mliki1, M. U. Höfer2
1Centre de Biotechnologie de Borj ce'dria, Laboratoire de Physiologie Mole'culaire de la Vigne;
B.P. 901, 2050Hammam-Lif, Tunisia 2RLP Agroscience GmbH, Alplanta-Institute for Plant Research, 67435 Neustadt a. d. Weinstraße, Germany
e-mail: samiabiotech@yahoo.fr Received May 17, 2011
Two subtracted cDNA libraries of grapevines (Vitis vinifera.L) under short term salt stress incubation were constructed using the suppression subtractive hybridization (SSH) method combined with the differential screening approach. The mRNA isolated from leaves of the salt-tolerant grapevine cultivar Razegui grown without stress was used as a "driver," and the corresponding mRNAs isolated after a short-term treatment 6 or 24h of salt stress were used as "testers." The differentially expressed cDNA fragments were identified by differential screening of these 2 libraries. During SSH procedure, each step was operated exactly according to the manual of the kit and the results were verified correct before following step. The libraries consisted of about 7000 recombinant clones, with the average size being of 500 bp, ranging from 100 bp to 900 bp. Using a PCR-select differential screening kit, 1000 recombinant clones were randomly chosen from the subtracted cDNA libraries and hybridized with forward, reverse subtracted and unsubtracted probes for two rounds. As a result, 848 positive clones were obtained. Sequencing of randomly selected clones from the differential screening revealed that most of transcripts over-expressed by salt stress have been reported as responsive to abiotic stress response.
One of the main limiting factors for plant agriculture in semi arid and arid area is the high salt levels in the soil and the poor quality of water used for irrigation. Tunisia as well as other Mediterranean countries is affected by this problem. In Tunisia, soils affected by salinity count for about 1.5 million of ha, representing near of10% of the total surface of the country and 25% of the arable land [1]. Depending on the plant species and the amount of salinity, plants will respond in different ways. When a certain tolerance level is reached, the plant will eventually die and when the plants in question are of high economic value, then the problem arises. This is creating the need for salt tolerant plants. Considerable researches have been devoted to quantify the salt and drought tolerance of various grapevine varieties either at the gene expression level [2, 3] or at the proteomic level [4, 5]. Results of those studies revealed that salt and drought stress induced massive and genome-wide changes in grapevine gene expression and protein synthesis. At the molecular level, gene products which are activated by salt stress belongs to several groups and are classified as functional proteins and regulatory proteins [6]. Functional proteins include water channel proteins, key enzymes for osmolyte biosynthesis, chaperones, LEA (late embryogenesis abundant) proteins, proteinases and detoxicating enzymes. Regulatory proteins include transcription factors, protein kinases, and phospholipases. Regulatory proteins are involved in the regulation of signal transduction and gene expression for stress tolerance [6, 7].
All these mechanisms are centered around the accumulation of certain differentially expressed gene products [8]. Signaling pathways can be divided into ABA-dependent pathways and ABA- independent pathways. These indicate the existence of complex regulatory mechanisms between perception of abiotic stress signals and gene expression [9]. A general assumption has been that stress up-regulated genes may be important for stress tolerance [10—13]. Several salt tolerant plants have been created through genetic manipulation of these genes [14, 15]. Techniques that evaluate global gene expression provide a powerful tool for the initial identification of important genes in the regulation of traits of economic importance. A variety of methods have been developped for this purpose and are now available, including differential display [16], Serial Analysis of Gene Expression (SAGE, [17]), Amplified Fragment Length Polymorphism (cDNA-AFLP, [18]), Suppressive Subtractive Hybridization (SSH, [19]). The latter technique was selected for this study. The SSH procedure includes a normalization step for mRNA abundance [20] particulary important for detecting low abundant transcripts. The SSH method exploits the suppression PCR effect, eliminating the need for physical separation of single and double stranded cDNAs [21]. The gene expression profiling approach has been well developped. Many studies using SSH technology have described changes in the transcriptome of many plants species, such as Arabi-dopsis [22], Populus [23], Cicer arietinum [24], maize
[25], alga [26] (Davidia involucrata) in response to abiotic stress. The large majority of research has been performed on herbacious species such as tobacco Ara-bidopsis, barely, rice, and tobacco, whereas few reports on the molecular response to salt stress exist in perennial woody plants. In this paper we focus on grapevine plant witch is of special interest because of its high economic value. The salt-tolerant variety Razegui has been chosen for this study to investigate the molecular mechanism of salt tolerance. Two subtracted cDNA libraries where constructed from stressed leaves. We differentially screened the library to identify genes up-regulated following salt-stress and sequenced some of the highly induced genes. Genes identified by this approach may serve not only as molecular markers for selecting salt tolerant plants but could be also used for future breeding programs against salt-stress.
MATERIALS AND METHODS
Plant material and stress treatment. Grapevine (Vitis vinifera L. var. Razegui) plants (6 months old) were grown hydroponically under an aerated nutrient solution, which contained Ca(NO3)2 =3.5 M, KNO3 = = 3 mM, NH4NO3 = 2 mM, K2HPO4 = 1.6 mM, MgSO4 = 1.5 mM, (NH4)2SO4 = 2.8 mM, FeSO4 = = 89 mM/EDTA (triplex 2) = 89.57 |M, MnCl2 = = 9 |M, ZnSO4 = 0.76 |M, CuSO4 = 0.70 |M, H3BO3 = 46.27 |M, KI = 0.43 mM and (NH4)6MO7O24 = 0.209 |M with pH 6.0. The electrical conductivity (ECe) of the nutrient solution was around 1.7 mS/cm-1. The hydroponic culture system was installed under controlled conditions in greenhouse (temperature: 24°C; relative humidity: 70%; light: 16h) to overcome the environmental interactions. Using hydroponic culture, salt tolerant variety is growing with or without 100mM NaCl in order to generate control and stressed plants as starting material for RNA purification. NaCl is added gradually to the culture medium (25 mM) each 3 days to a concentration of100 mM to avoid that the plants be shocked. In order to study the early response of the plants leaves, stems and roots from control and stressed samples were harvested separately after 6h (for cDNA mac-roarray probe) and 24h (for SSH library construction) of salt stress treatment and immediately frozen in liquid nitrogen and stored at -80° C until use.
Total RNA and mRNA isolation. Pooled leaves from nine plants per treatment were ground in liquid nitrogen. Total RNA was isolated according to [27]. For suppression subtractive hybridization, poly(A)+ RNA was purified from total RNA using streptavidin magnetic beads (New England Biolabs) according to the manufacturer's protocol. Quantity and quality of RNA samples were examined by spectrophotometry, gel electro-phoresis and RNA blot analysis, respectively.
Construction and screening of subtracted cDNA libraries. The suppressive subtractive hybridization cDNA templates were generated by reverse transcription of
2 |g of mRNA derived from control and stressed leaves.
Leaf specific SSH libraries were prepared using mRNA from 6 and 24 h salt-treated plants as tester and mRNA from control plants as driver and the PCR-select cDNA subtraction kit (Clontech, Takara Bio Europe) essentially according to the manufacturers protocol (PT1117-1.02/14/2002). 2 |L of nested SSH-PCR product of the forward subtraction were ligated by means of T/A-cloning using 50 ng of a linearised pT-PCR plasmid vector [28] and transformed into E. coli DH10B bacterial host. cDNA inserts from individual clones of SSH cDNA libraries (6 h and 24 h) were amplified in 96-well plate format by colony PCR from overnight Luria broth liquid cultures using flanking primers NPCR1 and NPCR2R as described (PCR-Select Differential Screening Kit manual, PT3138-1; Clontech, Takara Bio Europe). Ther-mocycling conditions were as follows: initial denatur-ation step at 94°C for 1.5 min, followed by 33 cycles at 94°C for 10 s, 68°C for 30 s, 72°C for 2 min and a final extension at 72°C for 5 min. All PCR products were analyzed by means of agarose gel electrophoresis to control quality and quantity.
Differential screening of subtracted cDNA libraries. In order to eliminate false-positive clones, a differential screening was performed. For the preparation of macro-arrays for screening purposes duplicates of 2 |L of colony PCR product were spotted onto Hy-bond-N+ nylon membranes (GE Healthcare Europe GmbH) with a split-pin 96-Multi-Blot Replica-tor(tm) (VP Scientific Inc.). 32P-labelled cDNA hybridisation probes prepared from subtracted SSH-PCR products and non subtracted ligation controls were generated using the HexaLabel DNA Labeling Kit (Fermentas) according to the manufacturer's instructions. After hybridisation, membranes were exposed to Kodak BioMax MS X-ray films (Sigma Aldrich) and differentially expressed candidate cDNA clones were then selected and re-arrayed into new 96-well microtiter plates.
Array hybridization and sequence analysis. The labeled probes were blocked by a
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