An isolated gene expressed in response to heat treatment in korean fir of abies genus
A Abies gene technology, applied in genomics, microbial measurement/testing, biochemical equipment and methods, etc.
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Embodiment 1
[0135] Example 1 Plant material and treatment
[0136] The seeds of Korean fir (Abies koreana Wilson) were collected from Halla Mountain (33°13-36’N, 126°12-57’E) in Jeju Island, South Korea. After breaking dormancy for 3 months at 4°C, seeds were sown in seedling trays with soil. Individual 1-year-old seedlings were transplanted into individual pots filled with the same soil. Plants are grown in a greenhouse under natural sunlight conditions. Heat stress treatment was performed under photoperiodic conditions (photon flux density of 180 μmol m -2 the s -1 ) were performed on 3 year old potted plants in a growth chamber set at 30°C. Three-year-old seedlings were exposed to normal growth conditions (22°C) and heat stress (30°C), then needles were harvested 21 days after heat treatment.
Embodiment 2
[0137] Example 2 Library Preparation and RNA Sequencing
[0138]RNA samples were extracted from needles of 21d heat-treated plants and control plants. Total RNA was isolated using TRIzol reagent according to the manufacturer's protocol (GibcoBRL, Cleveland, OH, USA). RNA quality and concentration were analyzed using 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). A total of 3 μg of RNA per sample was used for library construction using the Illumina Truseq RNA Sample Preparation Kit (Illumina, Inc. San Diego, CA, USA) according to the manufacturer's instructions. Briefly, mRNA is enriched using magnetic beads containing poly-T molecules. After purification, the enriched mRNA is fragmented into small fragments. First-strand cDNA was synthesized using random oligonucleotides and SuperScript II. DNA polymerase I and RNase H are then used to synthesize second-strand cDNA. Finally, these cDNA fragments were end-repaired and ligated with Illumina adapters. Librarie...
Embodiment 3
[0139] Example 3 De novo transcriptome assembly and annotation
[0140] Transcriptome assembly was done using Trinity software, which first combined reads with a certain length of overlap to form longer fragments without ambiguous bases, called contigs. The contigs were then joined by Trinity to generate sequences that could not be extended at either end. These sequences are called transcripts. Gene functions were annotated based on NCBI non-redundant protein sequences and GO. Transcript functional enrichment analysis using GO categories (Molecular Function, Biological Process and Cellular Component) was performed with the Blast2GO program (version 2.5.0).
[0141] The identification of embodiment 4DET
[0142] Transcript expression levels were calculated using the fragments per kb per million reads approach, which removes the effects of varying gene lengths and sequencing levels. To isolate DETs with two-fold higher or two-fold lower transcript expression between control ...
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