PubMed · 14084721
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C M SIMONS. 1963. LLOYD LIBRARY--100 YEARS.. https://pubmed.ncbi.nlm.nih.gov/14084721/
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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.
The synthetic lethal screen is a method of isolating novel mutants whose survival is dependent on a gene of interest. Combining the colony-color assay with a synthetic lethal screen offers a means to visually detect a mutant that depends on a plasmid for survival. Screening for synthetic lethals can be achieved in four steps. First, the gene of interest must be mutated in a strain harboring the ade2 ade3/ade8 mutations and producing white colonies. A plasmid containing the ADE3/ADE8 gene and the wild-type gene of interest must then be transformed into the strain, which results in red colonies with white sectors where the plasmid has been lost. A mutagenesis is then required to introduce random mutations into the yeast genome. Any cell with a mutation that causes dependence on the gene of interest for survival must maintain the plasmid; these cells will produce solid red colonies. Finally, the mutants are transformed with a library. The mutants containing complementing DNA are no longer dependent on the plasmid carrying the gene of interest and thus the synthetic lethals are identified by their red-white sectoring phenotype. The synthetic lethal gene can be identified by isolating and sequencing plasmid DNA.