ABSTRACT
The lily, a famous bulbous flower, is seriously affected by high temperatures, which affect their growth and production. To date, the signalling pathways and the molecular mechanisms related to heat response in Lilium have not been elucidated. In this study, a comparative transcriptome analysis was performed in an important thermo-tolerant flower, L. longiflorum, and a thermo-sensitive flower, L. distichum. Lily seedlings were first exposed to heat stress at 42°C for different lengths of time, and the optimal time-points (2 h and 24 h) were selected for RNA sequencing (RNA-seq). Approximately 66.51, 66.21, and 65.36 Mb clean reads were identified from three libraries of L. longiflorum (LL_CK, LL_T2h and LL_T24h, respectively) and 66.18, 66.03, and 65.16 Mb clean reads were obtained from three libraries of L. distichum (LD_CK, LD_T2h and LD_T24h, respectively) after rRNA removing. A total of 34,301 unigenes showed similarity to known proteins in the database NCBI non-redundant protein (NR), Swiss-Prot proteins, InterPro proteins, Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, 1,621 genes were differentially expressed in the overlapping libraries between LL_DEGs and LD_DEGs; of these genes, 352 DEGs were obviously upregulated in L. longiflorum and downregulated in L. distichum during heat stress, including 4-coumarate, CoA ligase (4CL), caffeoyl-CoA O-methyltransferase (CCoAOMT), peroxidase, pathogenesis-related protein 10 family genes (PR10s), 14-3-3 protein, leucine-rich repeat receptor-like protein kinase, and glycine-rich cell wall structural protein-like. These genes were mainly involved in metabolic pathways, phenylpropanoid biosynthesis, plant-pathogen interactions, plant hormone signal transduction, and kinase signalling pathways. Quantitative RT-PCR was performed to validate the expression profiling of these DEGs in RNA-seq data. Taken together, the results obtained in the present study provide a comprehensive sequence resource for the discovery of heat-resistance genes and reveal potential key components that are responsive to heat stress in lilies, which may help to elucidate the heat signal transcription networks and facilitate heat-resistance breeding in lily.
Subject(s)
Lilium/genetics , Lilium/physiology , Gene Expression Profiling , Gene Expression Regulation, Plant , Genes, Plant , Heat Shock Transcription Factors/genetics , Heat Shock Transcription Factors/physiology , Heat-Shock Response/genetics , Heat-Shock Response/physiology , Lilium/growth & development , Metabolic Networks and Pathways/genetics , Molecular Sequence Annotation , Multigene Family , Plant Proteins/genetics , Plant Proteins/physiology , RNA-Seq , Signal Transduction/genetics , Species Specificity , Thermotolerance/genetics , Thermotolerance/physiologyABSTRACT
The traditional primary culture methods of human normal epithelial cells have disadvantages of low activity of cultured cells,the low cultivated rate and complicated operation.To solve these problems,researchers made many studies on culture process of human normal primary epithelial cell.In this paper,we mainly introduce some methods used in separation and purification of human normal epithelial cells,such as tissue separation method,enzyme digestion separation method,mechanical brushing method,red blood cell lysis method,percoll layered medium density gradient separation method.We also review some methods used in the culture and subculture,including serum-free medium combined with low mass fraction serum culture method,mouse tail collagen coating method,and glass culture bottle combined with plastic culture dish culture method.The biological characteristics of human normal epithelial cells,the methods of immunocytochemical staining,trypan blue exclusion are described.Moreover,the factors affecting the aseptic operation,the conditions of the extracellular environment,the conditions of the extracellular environment during culture,the number of differential adhesion,and the selection and dosage of additives are summarized.
