低温诱导稻曲病菌菌核形成的转录组学分析

doi:10.3864/j.issn.0578-1752.2020.22.005

摘要/Abstract

摘要： 【目的】近40年来,稻曲病在世界各主要水稻栽培区均表现出发生规模不断扩大、严重程度不断增加的趋势,并逐渐发展成为水稻主要病害之一。前期对低温诱导初期的稻曲球进行切片,发现稻曲球内含有大量隐含菌核。本研究通过转录组高通量测序鉴定低温诱导稻曲病菌（Villosiclava virens）菌核形成的潜在调控基因,阐释菌核形成的分子机制,为揭示稻曲病发生规律及有效防治打下基础。【方法】利用高通量测序技术,对低温诱导处理的稻曲球进行转录组测序（对照组：TL911_1、TL911_2、TL911_3;低温处理组：FH1016_1、FH1016_2、FH1016_3）。以稻曲病菌基因组（UV-8b）作为参考基因组进行序列比对,利用FPKM法计算基因表达量,设定参数（|log₂ fold change|≥1且q-value≤0.05）筛选差异表达基因。结合基因差异表达分析、基因家族分析和富集分析（Gene Ontology/KEGG Pathway）,鉴定稻曲病菌菌核形成关键基因,并利用实时荧光定量PCR（qRT-PCR）技术对其表达量进行验证。【结果】转录组测序共获得59.78 G高质量数据,其中,近93.2%的数据能够比对到稻曲病菌基因组。数据分析共鉴定到8 426个基因存在不同程度表达,占总体基因的97.13%。与对照相比,低温处理可诱导793个基因显著差异表达,分别有398和395个基因表现为上调、下调表达,随机挑选6个基因进行qRT-PCR验证,试验结果与转录组分析一致。在差异表达基因中,共注释到180个（22.7%）基因家族,其中61.67%的基因家族表现为上调表达,主要包括MFS转运蛋白、糖转运蛋白、锌指转录因子等。GO富集分析发现,差异表达基因显著富集于碳水化合物代谢、氧化还原过程、氧化还原酶活性等。KEGG分析发现,差异表达基因显著富集于次生代谢产物生物合成、淀粉蔗糖代谢、糖酵解/糖异生等代谢通路,暗示营养物质代谢和能量代谢途径相关基因表达对于低温诱导菌核形成至关重要。【结论】低温可诱导稻曲病菌菌核形成。低温使机体内部处于氧化应激状态,可通过信号转导途径放大,该过程由多个基因参与并调控多个基因家族成员,最终促使跨膜运输、细胞形态、生物合成等基因的上调表达,使得在形成菌核过程中蛋白表达活跃,达到合成细胞及物质的高峰期,进而促进菌核形成。

关键词: 稻曲病菌, 菌核, 转录组, 低温胁迫, 氧化应激反应, 差异表达基因

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)

吕楚阳,邓平川,张晓丽,孙钰超,梁五生,胡东维. 低温诱导稻曲病菌菌核形成的转录组学分析[J]. 中国农业科学, 2020, 53(22): 4571-4583.

LÜ ChuYang,DENG PingChuan,ZHANG XiaoLi,SUN YuChao,LIANG WuSheng,HU DongWei. Transcriptomic Analysis of Sclerotia Formation Induced by Low Temperature in Villosiclava virens[J]. Scientia Agricultura Sinica, 2020, 53(22): 4571-4583.

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序号No.	蛋白名称 Protein name	蛋白ID Protein ID	引物序列 Primer sequence (5′-3′)	扩增长度 Amplicon size (bp)
1	Protein rds1	KDB11131.1	F: TCCAACGTGCGGGAATACA	164
1	Protein rds1	KDB11131.1	R: CCAAACTGGCGGAAAATC	164
2	Biotrophy-associated secreted protein 2	KDB17523.1	F: GCCTCAGCAACACCGACT	220
2	Biotrophy-associated secreted protein 2	KDB17523.1	R: GCCTTTACCTCGTCCGCC	220
3	Glycoside hydrolase	KDB17641.1	F: TCCTCGCCACCATCTCGT	178
3	Glycoside hydrolase	KDB17641.1	R: CGCCTCATCGCCCTCAAC	178
4	Cytosolic phospholipase A2 zeta	KDB15918.1	F: ATGGGCGTCTTTGGGAGCG	165
4	Cytosolic phospholipase A2 zeta	KDB15918.1	R: GGGAATTGTGGCGGGATCT	165
5	CRE-TRX-1 protein	KDB18233.1	F: GCCAAATCCCGACAAAAG	142
5	CRE-TRX-1 protein	KDB18233.1	R: GGCGGCGTAGTCACCATA	142
6	Methylenetetrahydrofolate reductase 1	KDB13169.1	F: TCCTCGCCACCATCTCGTC	179
6	Methylenetetrahydrofolate reductase 1	KDB13169.1	R: CCGCCTCATCGCCCTCAAC	179
7	α-tubulin	KDB12764.1	F: GCTCTCGTGCTTGCTCTTGG	144
7	α-tubulin	KDB12764.1	R: ATCACTTCGTCCTTGCGTTT	144

样品 Sample	总数据 Total reads	比对数据 Mapped reads	比对率 Mapped ratio (%)	有效序列 Clean bases (G)	GC含量 GC content (%)	Q30 (%)
TL911_1	59349736	55846678	94.10	8.90	57.15	93.51
TL911_2	59738802	55976056	93.70	8.96	56.95	93.09
TL911_3	72656794	68033430	93.64	10.90	56.85	93.01
FH1016_1	59387684	54809829	92.29	8.91	57.09	92.45
FH1016_2	63542726	58890927	92.68	9.53	57.25	93.53
FH1016_3	83852442	77646536	92.60	12.58	57.22	93.53
总计Total	398528184	371203456	-	59.78	-	-

途径ID Pathway ID	途径描述 Description of pathway	DEG数量 DEGs number	上调数量 Up-regulated number	下调数量 Down-regulated number	P值 P-value
fgr01110	次生代谢产物生物合成Biosynthesis of secondary metabolites	174	119	55	0.000456851
fgr00500	淀粉和蔗糖代谢Starch and sucrose metabolism	25	20	5	0.001216228
fgr00010	糖酵解/糖异生Glycolysis/Gluconeogenesis	23	17	6	0.002826092
fgr01200	碳代谢Carbon metabolism	64	42	22	0.003358729
fgr00680	甲烷代谢Methane metabolism	15	10	5	0.004476103
fgr01130	抗生素的生物合成Biosynthesis of antibiotics	128	86	42	0.004815518
fgr00250	丙氨酸、天冬氨酸和谷氨酸代谢Alanine, aspartate and glutamate metabolism	19	13	6	0.006041910
fgr00520	氨基糖和核苷酸糖代谢Amino sugar and nucleotide sugar metabolism	27	15	12	0.006396843