Abstract:

【Objective】In the past 40 years, the occurrence scale and severity of rice false smut have been increasing in all major rice cultivation areas in the world, and it has gradually developed into a major rice disease. The previous study found that there were a lot of internal growth sclerotia induced by low temperature at the initial stage. The objective of this study is to screen out the potential regulatory genes involved in sclerotia development induced by low temperature through high-throughput sequencing, explain the molecular mechanism of sclerotia formation, and to lay a foundation for revealing the occurrence rule of rice false smut and preventing its epidemic more effectively.【Method】The high-throughput sequencing was used to analyze the transcriptome data of rice false smut balls by low-temperature induction (Control group: TL911_1, TL911_2, TL911_3. Low-temperature treatment group: FH1016_1, FH1016_2, FH1016_3). The genome of Villosiclava virens (UV-8b) was used as a reference to align the sequences. The gene expression level was calculated in the term of Fragments per Kilobase of transcript per Million fragments (FPKM). Compared with the database, the differentially expressed genes (DEGs) were screened by parameters (|log₂ fold change|≥1 and q-value≤0.05). Combined with gene differential expression analysis, gene family analysis and function annotation (Gene Ontology/KEGG Pathway), the key genes of sclerotia formation in V. virens were identified. qRT-PCR was performed to validate the expression of screened genes associated with sclerotia development.【Result】A total of 59.78 G high-quality data were obtained by transcriptome sequencing, of which nearly 93.2% of the data could be mapped to the genome of V. virens. Data analysis showed that 8 426 genes were expressed in different degrees, accounting for 97.13% of the total genes. Compared with the control, 793 genes were significantly differentially expressed by low-temperature treatment, 398 and 395 genes were up- and down-regulated, respectively. The 6 genes were randomly selected and verified by qRT-PCR. The result was consistent with the transcriptome analysis. Among the DEGs, 180 gene families (22.7%) were annotated, among which 61.67% were up-regulated, including MFS transporter, sugar transporter, zinc finger TF, etc. GO enrichment analysis showed that the DEGs were significantly enriched in carbohydrate metabolism process, oxidation-reduction process, oxidoreductase activity, etc. KEGG analysis showed that the DEGs were significantly enriched in biosynthesis of secondary metabolites, starch and sucrose metabolism, glycolysis/gluconeogenesis and other metabolic pathways, suggesting that the expression of genes related to nutrient metabolism and energy metabolism pathway is essential for the formation of sclerotia induced by low temperature.【Conclusion】Low-temperature stress can induce the formation of sclerotia, which makes the fungi in an oxidative stress state. Through the amplification of signal transduction pathway, the whole process involves multiple genes and regulates multiple gene family members, which ultimately promotes the up-regulated expression of genes such as transmembrane transport, cell morphology and biosynthesis, so that protein expression is active in the process of forming sclerotia, reaching the peak of synthetic cells and substances, and then promoting the formation of sclerotia.

Key words: Villosiclava virens, sclerotia, transcriptome, low-temperature stress, oxidative stress response, differentially expressed gene (DEG)