LOCUS       FC070573                 627 bp    mRNA    linear   EST 14-JUN-2011
DEFINITION  sT7aVVM028C06029 VVM Vitis vinifera cDNA clone VVM028C06029 5'
            similar to dbj_BAE91898.1 tobacco fibrillarin homolog [Nicotiana
            tabacum]. Expect = 3e-87, mRNA sequence.
ACCESSION   FC070573
VERSION     FC070573.1
DBLINK      BioSample: SAMN00164509
KEYWORDS    EST.
SOURCE      Vitis vinifera (wine grape)
  ORGANISM  Vitis vinifera
            Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
            Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae;
            Pentapetalae; rosids; Vitales; Vitaceae; Viteae; Vitis.
REFERENCE   1  (bases 1 to 627)
  AUTHORS   Tillett,R.L., Ergul,A., Albion,R.L., Schlauch,K.A., Cramer,G.R. and
            Cushman,J.C.
  TITLE     Identification of tissue-specific, abiotic stress-responsive gene
            expression patterns in wine grape (Vitis vinifera L.) based on
            curation and mining of large-scale EST data sets
  JOURNAL   BMC Plant Biol. 11 (1), 86 (2011)
   PUBMED   21592389
COMMENT     Contact: Cushman JC
            Department of Biochemistry
            University of Nevada
            MS200, Reno, NV 89557-0014, USA
            Tel: 775-784-1918
            Fax: 775-784-1650
            Email: jcushman@unr.edu
            PCR PRimers
            FORWARD: T7 (TAATACGACTCACTATAGGG)
            BACKWARD: T3 (AATTAACCCTCACTAAAGGG)
            Plate: 028  row: C  column: 06
            Seq primer: T7 (TAATACGACTCACTATAGGG)
            POLYA=No.
FEATURES             Location/Qualifiers
     source          1..627
                     /organism="Vitis vinifera"
                     /mol_type="mRNA"
                     /cultivar="Cabernet Sauvignon"
                     /db_xref="taxon:29760"
                     /clone="VVM028C06029"
                     /tissue_type="roots"
                     /clone_lib="SAMN00164509 VVM"
                     /dev_stage="10 cm high plants grown in Magenta boxes"
                     /note="Vector: Lambda Uni-Zap XR, Bluescript SK-; Site_1:
                     EcoRI; Site_2: XhoI; Vitis vinifera roots grown in
                     Murashige and Skoog (1962) macro and micronutrients with
                     Gamborg's B5 vitamins (Gamborg et al., 1968) supplemented
                     with 1.5% sucrose, 1% agar under control conditions as
                     well as subjected to cold (0-1.5oC), water-deficit, and
                     150 mM NaCl stress for 2, 4, 6 h. cDNA Library was
                     constructed using total RNA from cold, drought, 150 mM
                     NaCl stressed and well watered Vitis vinifera roots. Total
                     RNAs from different treatments were extracted and equal
                     amounts were pooled before mRNA selection. Poly(A)+mRNA
                     was isolated from total RNA using the Oligotex Direct mRNA
                     kit (Qiagen). cDNA synthesis was conducted by coverting
                     poly(A)+mRNA to double - stranded cDNA by using primer:
                     5'-AACTGGAAGAATTCGCGGCCGCTCGCATTTTTTTTTTTTTTTTTTV-3'
                     (V=A,C,G) and Superscript III (Invitrogen). Double
                     stranded cDNAs were size selected (more than 600 bp),
                     adaptored with EcoRI adaptors (aattccgttgctgtcg - Promega
                     # C1291) and digested with NotI. The cDNAs were then
                     directionally cloned into EcoRI-NotI digested pBluescript
                     II SK+ phagemid vector (Stratagene). The total number of
                     white colony forming units (cfu) before amplification was
                     3x106. Blue colonies (empty vectors) were less than 10%.
                     Purified plasmid DNA from the primary library was
                     converted to single-stranded circles and used as a
                     template for PCR amplification using the T7 and T3 priming
                     sites flanking the cloned cDNA inserts as previously
                     described (Bonaldo M, Lennon G, and Soares MB (1996)
                     Normalization and subtraction: two approaches to
                     facilitate gene discovery. Genome Research 9:791-806). The
                     purified PCR products, representing the entire cloned cDNA
                     population, were used as a driver for normalization.
                     Hybridization between the single-stranded library (50ng)
                     and the PCR products (500ng) was carried out for 44 hours
                     at 30oC. Unhybridized single-stranded DNA circles were
                     separated from hybridized DNA rendered partially
                     double-stranded and electroporated into DH10B cells to
                     generate the normalized library. The total number of
                     clones with insert was: 1.6x106 cfu. Background of empty
                     clones was less than 10%."
BASE COUNT          145 a          110 c          227 g          145 t
ORIGIN      
        1 acccttagca gagatgagac ctccagcagg aagaggccgt ggaggtggcg gtttcggagg
       61 tggcggtttc agaggcagag gcgatggagg aaggggcaga ggcagaggag ggtttggtgg
      121 aagaggtggc agtgacatga acagccgggg tggtggtcgt ggcgaccgtg gtggtcgtgg
      181 tggtcgtggc ggcgggagag gccgtggtgg acgcggaggt ggcggaatga aaggtggaag
      241 cagggttgta gttgagcccc ataggcatga gggggtgttc attgctaagg gcaaagaaga
      301 tgcacttgtt actaagaata tggtccccgg agaagccgtc tacaatgaga aaagaatctc
      361 tgttcagaat gaagatggaa gcaaaattga atacagggtt tggaacccct tccgttcaaa
      421 gttggctgct gccattcttg gtggtgttga tgagatctgg attaaacctg gtgctcgagt
      481 tctctacctt ggtgctgcct cagggaccac tgtttcccat gtctcggatg ttgttggccc
      541 tactggagtg gtttatgcag tggaattttc tcacagaagt ggtagggact tggttaacat
      601 ggcaaagaag cggacaaatg ttattcc
//