Research On The Expressed Sequence Tag Of Olives

835 words - 4 pages

Olive, or scientific name Olea europaea L which belongs to Oleacea family is a valuable plant species where it can produce oil and table olives. Gene at certain tissue in species will be expressed where cDNA are sequenced to produce expressed sequence tag (EST) library. EST database is beneficial as it allows new gene discovery, marker discovery, gene mapping, and functional studies to be carried out. This research has contributed EST library of 2304 clone sequences from the young olive leaf and 1536 clone sequences from the immature olive fruit for Turkish olive cultivar Gemlik. Good quality ESTs are used to further analysed by using Phred-Phrap and Contig Assembly Program 3 (CAP3) software. They were then submitted to GenBank (dbEST). BLAST and BLAST2GO are conducted to perform annotation of the sequences.

Total RNA was isolated from fresh young leaves and immature olive fruits with RNeasy PlantMiniprep kit (Qiagen). Total RNA was then converted into 1.5 μg and 3μg mRNA respectively by purification, using the Oligotex mRNA Mini Kit (Qiagen). DNA libraries were then constructed with the CloneMiner cDNA Library Construction Kit (Invitrogen). cDNA was cloned into pDONR222 vector and transformed into E.coli strain DH5 (Invitrogen). The cDNA library was plated onto LB-kanamycin agar medium. Colonies which were grown individually were then picked into 384-well plates with SOB medium to be inoculated overnight. They were then added with glycerol. The library was stored at −80˚C. The above steps were done separately for the two cDNA library.
Sixty clones randomly selected. Alkaline lysis method was used to isolate plasmid DNA from the clones selected previously. Bgl1701 was then used to digest the DNA.
Polymerase chain reaction (PCR) was carried out to amplify the clones to create library. 3840 clones were selected randomly to be used as PCR template. M13 universal primer was used as primer in PCR. Automated sequencing was performed using the ABI 3730 capillary sequencer (PE Applied Biosystems) to obtain the sequences of clones.
Low-quality EST sequences were removed by using Phred software. EST sequences with vector sequence were edited using Phrap “cross-match” application. Contig Assembly Program 3 (Cap3) was used to assemble the sequences obtained from sequencing for analysis while Consed/Autofinish software was used to control the sequence assembly. All sequences were assembled separately into contigs. BLAST of sequences was conducted to determine the gene homology in order to connect their functions. Unique sequences were analysed for biological characteristics as well as functional annotation using program BLAST2GO. New genes can then be...

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