RESUMO
Long intergenic noncoding RNAs (lincRNAs), which possess diverse features such as remodeling chromatin and genome architecture, RNA stabilization, and genome architecture, are important regulatory factors in plant genomes. They serve to fine-tune the expression of neighboring genes. Here, we describe a procedure of discovery, identification, and functional characterization of plant lincRNAs after virus infection. From high-throughput RNA-Seq transcriptome analysis, the noncoding RNA transcripts with significant fold changes (upregulation or downregulation) will be discovered and identified. The lincRNA of interest will be further confirmed and validated using rapid amplification of cDNA ends (RACE). In addition, functional characterization of the lincRNA will be followed up through overexpression and knockdown strategies.
Assuntos
Arabidopsis/genética , Arabidopsis/virologia , DNA Intergênico/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , RNA Longo não Codificante/genética , RNA de Plantas/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA , TranscriptomaRESUMO
KEY MESSAGE: A long intergenic noncoding RNA LINC - AP2 is upregulated and negatively correlated with AP2 gene expression with Turnip crinkle virus infection in Arabidopsis. Plant vegetative growth and floral reproductive structure were severely retarded and distorted in Turnip crinkle virus (TCV)-infected Arabidopsis thaliana. Compared to mock-inoculated plants, the stamen filaments were shorter in flowers of TCV-infected plants. However, TCV-infected plants can still produce normal seeds through artificial pollination, indicating both its pollen and stigma were biologically functional. From our high-throughput RNA-Seq transcriptome analysis, a floral structure-related APETALA2 (AP2) gene was found to be downregulated and its neighboring long intergenic noncoding RNAs (lincRNA), At4NC069370 (named LINC-AP2 in this study), were upregulated significantly in TCV-infected plants. This LINC-AP2 was further confirmed for its existence using 5'RACE technology. LINC-AP2 overexpression (LINC-AP2 OE) transgenic Arabidopsis plants were generated to compare with TCV-infected WT plants. TCV-infected LINC-AP2 OE plants which contained lower AP2 gene expression displayed more severe symptoms (including floral structure distortion) and higher TCV-CP gene transcript and coat protein levels. Furthermore, compared to TCV-infected WT plants, TCV-infected ap2 mutant plants failed to open their flower buds and displayed more severe viral symptoms. In conclusion, upregulation of LINC-AP2 is negatively correlated with AP2 gene expression with TCV infection in Arabidopsis.
