关键词: 红橘, 链格孢菌橘致病型, 转录组, 差异表达基因

Abstract:

【Objective】The objective of this study is to reveal the gene expression pattern at transcriptional level of tangerine (Citrus reticulata Blanco, cv. Hongjv) after inoculated with the brown spot pathogen (Alternaria alternata tangerine pathotype), and to identify the key genes of tangerine responding to the pathogen infection.【Method】Detached leaves of tangerine were inoculated with A. alternata tangerine pathotype for 28 h and used for RNA extraction and transcriptome sequencing. Sequencing results were verified by quantitative real-time PCR (qRT-PCR). Differentially expressed genes (DEGs) were screened with |log₂ fold change|≥1, q-value≤0.01 as threshold and clean reads were compared with genome of sweet orange (Citrus sinensis). GO database was used to perform functional classification of DEGs, KEGG was used to analyze metabolic pathways, and MapMan software was used to analyze the expression changes of genes related to biological stress signaling pathways.【Result】A large number of DEGs related to stress were produced at 28 h post inoculation, 5 173 up-regulated and 6 555 down-regulated genes were obtained. GO functional classification showed that the DEGs were mainly related to protein binding, membrane and oxidation-reduction process. KEGG enrichment and MapMan software analysis revealed that the basic metabolism of tangerine was severely damaged during the stress of A. alternata tangerine pathotype. The expression of multiple genes in host defense-related plant hormone signal transduction pathways such as ethylene (ET), salicylic acid (SA) and auxin (AUX) varied to some extent, of which ET plays a key role. The three members of the ET receptor ETR were activated to different degrees, among which downstream kinases and ET response factors were up-regulated, whereas most key signal genes of AUX, most members of AUX response factor ARF and genes in SA synthesis pathway were all down-regulated. Meanwhile, genes related to flavonol, anthocyanins, terpenoids and alkaloid synthesis were drastically changed by the fungus. Most genes of terpenoid synthesis were down-regulated, while the number and expression trend of up-regulated genes related to flavonoid synthesis were stronger than those of the down-regulated genes. Glucosinolate genes with anti-insect and antibacterial effects were up-regulated. Furthermore, a large number of transcription factors involved in stress resistance processes such as WRKY, bZIP, ERF, MYB and NAC were induced and activated. Most members of WRKY and bZIP transcription factors were positively regulated by the fungus, and the expressions of more than 50% of the ERF family genes were up-regulated. Under the regulation of transcription factors, PTI and ETI response genes such as receptor kinases, R proteins and NBS anti-disease proteins were largely expressed, multiple resistance PR genes were up-regulated and the expressions of 22 POD genes of antioxidant protective enzyme systems were largely activated by reactive oxygen species. The above results indicated that A. alternata tangerine pathotype infection had a great effect on the physiological state of citrus plants. To validate the findings, 19 DEGs related to anti-disease were selected for further analyses using qRT-PCR. The result was fairly consistent with that of transcriptional sequencing.【Conclusion】The DEGs and the significantly up-regulated expression genes obtained in C. reticulata Blanco, cv. Hongjv were mainly involved in the metabolic process, the response to stress and the transcriptional regulation. The synergistic regulation of these genes is an important mechanism of defense reaction to A. alternata tangerine pathotype.

Key words: Citrus reticulata Blanco, cv. Hongjv, Alternaria alternata tangerine pathotype, transcriptome, differentially expressed gene (DEG)