PubMed · 13191968
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1954-09-18. LIBRARY.. https://pubmed.ncbi.nlm.nih.gov/13191968/
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To systematically analyze the gene function of Dalbergia odorifera, the seedlings of D. odorifera were treated with low-temperature stress for 6 h. Total RNA was extracted from a mixture of seedling roots, stems, and leaves, and a low-temperature-induced D. odorifera yeast cDNA expression library was constructed. The library volume was 1.032 × 108 CFU, and the PCR (Polymerase Chain Reaction) identification of the library bacterial fluid showed that the amplification was around 1000 bp, with a single randomly distributed band, indicating that the library had been recombinantly inserted into the pYES2 vector. The GO (Gene Ontology) analysis showed that the library genes were mainly involved in metabolic and stress signaling pathways. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis showed that the genes were primarily related to energy and metabolic pathways. Twenty-one genes were screened or obtained at -20°C for low-temperature tolerance. In addition, the organ expression profiles of the candidate genes were analyzed based on RNA-seq data, and the expression profiles of the candidate genes under low-temperature stress were also examined. The construction of the yeast library provides genetic resources for the analysis of the mechanism of low-temperature tolerance of D. odorifera, which is important for comprehending and utilizing the genetic resources of D. odorifera.
RNA sequencing (RNA-seq) methodologies allow the discovery of novel variants and transcripts. These comprise three general steps: (1) capture of RNA species of interest, (2) conversion of RNA to complementary DNA (cDNA), and (3) modification of cDNA to fit the sequencing platform. Here we describe four different library preparation protocols for short-read sequencing: cDNA synthesis with poly(A) selection, library preparation with ribosomal depletion, and cDNA synthesis with SMART® (Switching Mechanism at 5' end of RNA Template) technology for low and Pico inputs.
Retroelements (REs) occupy up to 40% of the human genome. Newly integrated REs can change the pattern of expression of pre-existing host genes and therefore might play a significant role in evolution. In particular, human- and primate-specific REs could affect the divergence of the Hominoidea superfamily. A comparative genome-wide analysis of RE sites of integration, neighboring genes, and their regulatory interplay in human and ape genomes would be of help in understanding the impact of REs on evolution and genome regulation. We have developed a technique for the genome-wide comparison of the integrations of transposable elements in genomic DNAs of closely related species. The technique called targeted genome differences analysis (TGDA) is also useful for the detection of deletion/insertion polymorphisms of REs. The technique is based on an enhanced version of subtractive hybridization and does not require preliminary knowledge of the genome sequences under comparison. In this report, we describe its application to the detection and analysis of human specific L1 integrations and their polymorphisms. We obtained a library highly enriched in human-specific L1 insertions and identified 24 such new insertions. Many of these insertions are polymorphic in human populations. The total number of human-specific L1 inserts was estimated to be approximately 4000. The results suggest that TGDA is a universal method that can be successfully used for the detection of evolutionary and polymorphic markers in any closely related genomes.