CONTIGS and ESTs from Gibberella zeae (anamorph Fusarium graminearum)
A description of these ESTs has been published (Fungal Genetics and Biology 38:187-197).. The work is summarized below.
LINKS to ESTs and Contigs:
Click HERE for a summary of ESTs against the nr database (includes all of the contigs).
To view contigs and the ESTs that comprise them, Click HERE for contigs. Click HERE for singletons.
Analysis of Expressed Sequence Tags from Gibberella zeae (anamorph Fusarium
Frances Trail, Jin-Rong Xu†, Phillip San Miguel†, Robert G. Halgren* and H. Corby Kistler‡
Departments of Plant Biology and Plant Pathology, and *Genomics Technology
Support Facility, Michigan State University, East Lansing, MI 48824.
† Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907.
‡ USDA ARS Cereal Disease Laboratory, and Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108.
You may contact any of us, Frances Trail maintains this web page. Her specifiecs are: Tel: 517-432-2939. Fax: 517-3535-1926. E-mail: email@example.com.
We have generated 7996 ESTs from three cDNA libraries. Two libraries were generated from carbon- (C-) and nitrogen- (N-) starved mycelia and one library was generated from cultures of maturing perithecia (P). In other fungal pathogens, starvation conditions have been shown to act as cues to induce infection-related gene expression. To assign putative function to cDNAs, sequences were initially assembled using StackPack. The estimated total number of genes identified from the three EST databases wass 2110: 1088 contigs and 1022 singleton sequences. These 2110 sequences were compared to a yeast protein sequence reference set and to the GenBank non-redundant database using BLASTX.
EST numbers: Perithecium derived ESTs begin with a four digit number 1601-1650. C-starved begin with FgrC. N-starved begin with FgrN and Fgr. All FgrS sequences are from libraries prepared with wheat heads inculated with Fusarium graminearum. There are many plant sequences amongst the fungal sequences in the FgrS ESTs. These sequences will be published elsewhere.
Culture conditions and library preparation.
G. zeae lineage 7 strain PH-1 (NRRL 31084; Trail and Common, 2000) was used for construction of all cDNA libraries and maintained as soil stocks at -20 C. To construct the library from sexually sporulating cultures, nearly synchronous lawns of perithecia were produced on carrot agar as previously described (Klittich and Leslie, 1988). After 2-4 d of growth on carrot agar in 60 mm Petri dishes, the mycelium reached the perimeter of the plate. Perithecia were induced from vegetative hyphae at this stage with the addition of 1 ml of 2.5 % aqueous Tween 60 (Sigma Company, St. Louis, Missouri). Developing perithecia, 6 days post induction, were gently scraped from the surface of carrot agar cultures with a sterile scalpel and immediately frozen at -80oC. Tissue was subsequently lyophilized and total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) following manufacturer’s directions. From total RNA, mRNA was prepared using PolyATract mRNA Isolation System II (Promega, Madison, WI) according to manufacturers directions. The perithecium cDNA library was constructed using the SuperScript Lambda System with ZipLox, Not I-Sal I Arms (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. The plasmid was excised in the host strain DH10B(ZIP).
For libraries from C- and N-starved cultures, PH-1 mycelia were collected from 1 L of 2-day-old culture grown in complete medium (CM, Correll et al., 1987), rinsed with sterile distilled water, and transferred into 1 L liquid minimal medium (MM, Correll et al., 1987) lacking a carbon- or nitrogen- source, respectively. After incubation for an additional 24 h with shaking at 150 rpm, mycelia starved for carbon or nitrogen were collected by filtration through 1 layer of Miracloth (Calbiochem, LaJolla, CA) and frozen at -80°C. Total RNA was extracted as above and mRNA was purified with the Oligotex mRNA isolation kit (Qiagen Inc., La Jolla, CA). The cDNA libraries were synthesized in the vector Uni-ZapII with a cDNA synthesis kit (Stratagene, LaJolla, CA). The cDNA inserts were directionally cloned between the EcoRI and XhoI sites of pBluescript (with the 3’ polyA tails close to the XhoI site). Both C- and N- cDNA libraries were excised according to manufacturer's directions. For all libraries, individual white colonies were transferred by a Q-Pix robotic workstation (Genetix, UK) to 384-well microplates and preserved at -80oC.