番茄青枯病抗性相关miRNA的鉴定与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1241

摘要/Abstract

摘要：

为理解番茄（Solanum lycopersicum）青枯病抗性响应miRNA与靶基因间的调控关系，对抗、感番茄自交系接种青枯菌（Ralstonia solanacearum）前后进行小RNA测序。结果在8个样本中共获得112.76 M高质量数据，检测到336个miRNA，包括193个新miRNA。其中，31个差异表达miRNA靶向调节575个基因的表达。556个靶基因被注释到防御反应、植病互作、植物激素信号转导等代谢途径中。启动子除典型的转录起始TATA-box和CAAT-box，及与生物胁迫相关的W-box和TC-rich repeats，还存在激素、光、非生物胁迫、伤等响应元件。RT-qPCR验证发现6对miRNA-targets具有正-负、负-正和负-负3种番茄青枯病应答模式。这些结果初步揭示miRNA可以通过靶向基因表达响应番茄青枯病。

关键词: 番茄, 青枯菌, miRNA, 靶基因, 表达调控

Abstract:

To elucidate the regulatory relationship of miRNA-targets in the bacterial wilt response of tomato（Solanum lycopersicum）, sRNA sequencing was performed in resistant and susceptible tomato inbred lines with Ralstonia solanacearum vs mock inoculation. In all, 112.76 M high-quality reads were generated in eight libraries. A total of 336 miRNA including 193 novel miRNAs were identified. Among them, 31 differentially expressed miRNAs targeted the regulatory expressions of 575 genes. In total, 556 target genes were annotated in metabolic pathways such as defense response, plant-pathogen interaction, and plant hormone signal transduction. In addition to typical TATA-box and CAAT-box with the transcriptional initiation role, as well as W-box and TC-rich repeats related to biotic stress, there were also hormone, light, abiotic stress, wound and other response elements in the promoters. RT-qPCR confirmed that six pairs of miRNA-targets had three bacterial wilt response patterns in tomato, i.e., positive-negative, negative-positive and negative-negative. These results preliminarily revealed the miRNAs may respond to the bacterial wilt disease by target gene expression in tomato.

Key words: tomato, Ralstonia solanacearum, miRNA, target gene, expression regulation

史建磊, 宰文珊, 苏世闻, 付存念, 熊自立. 番茄青枯病抗性相关miRNA的鉴定与表达分析[J]. 生物技术通报, 2023, 39(5): 233-242.

SHI Jian-lei, ZAI Wen-shan, SU Shi-wen, FU Cun-nian, XIONG Zi-li. Identification and Expression Analysis of miRNA Related to Bacterial Wilt Resistance in Tomato[J]. Biotechnology Bulletin, 2023, 39(5): 233-242.

图/表 9

表1 RT-qPCR引物

Table 1 Primers used for RT-qPCR

基因 Gene	序列 Sequence（5'-3'）	引物 Primer（5'-3'）
SlU6	GTCCCTTCGGGGACATCCGATAAAATTGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATGACACGCACAAATCGAGAAATGGTCCAAAATTTT	F: GGGACATCCGATAAAATTGG R: TTGGACCATTTCTCGATTTGT
novel_miR_13	GGCGGAUGUAGCCAAGUGGA	RT: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCCACTT F: ACACTCCAGCTGGGGGCGGATGTAGCC
sly-miR172c	AGAATCTTGATGATGCTGCAG	RT: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTGCAGC F: ACACTCCAGCTGGGAGAATCTTGATGAT
sly-miR172d	GGAATCTTGATGATGCTGCAG	RT: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTGCAGC F: ACACTCCAGCTGGGGGAATCTTGATGAT
sly-miR396a-5p	TTCCACAGCTTTCTTGAACTG	RT: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGTTCAA F: ACACTCCAGCTGGGTTCCACAGCTTTC
		All R: TGGTGTCGTGGAGTCG

表2 番茄8个样本小RNA测序数据统计

Table 2 Statistics of sRNA sequencing reads in the eight tomato libraries

样本Library	净读数Clean reads/M	净碱基数Clean bases/Gb	GC含量GC content/%	Q30/%	无注释Unannotated	比对读数Mapped reads
RC02	14.31	0.32	41.86	95.26	10.81	7.08
RC14	16.69	0.38	42.34	95.26	11.86	7.68
RT06	12.93	0.30	42.68	95.61	8.85	5.72
RT18	10.84	0.24	41.95	95.57	7.92	5.11
SC04	14.57	0.33	42.10	95.68	10.22	7.31
SC16	16.54	0.37	43.15	95.75	10.79	7.77
ST08	11.32	0.26	42.76	95.55	7.68	5.14
ST20	15.56	0.35	41.62	95.77	11.52	7.75

表3 各样本已知和新miRNA统计

Table 3 Statistics of known and new miRNA in each lib-rary

样本 Library	已知miRNA Known miRNA	新miRNA Novel miRNA	总数 Total	新miRNA百分率 Novel miRNA percentage/%
RC02	134	193	327	59.02
RC14	138	193	331	58.31
RT06	136	192	328	58.54
RT18	132	190	322	59.01
SC04	137	193	330	58.48
SC16	136	193	329	58.66
ST08	134	193	327	59.02
ST20	136	193	329	58.66
Total	143	193	336	57.44

图1 抗、感番茄中的差异表达miRNA Up和Down分别表示基因上调和下调表达

Fig. 1 Differentially expressed miRNA in resistance and susceptible tomato lines Up and Down indicate up-regulated and down-regulated expression, respectively

表4 miRNA靶基因数目统计

Table 4 Statistics of miRNA targets

类型 Type	所有miRNA All miRNA	具有靶基因的miRNA miRNA with targets	靶基因 Target genes
Known miRNA	143	134	2 268
Novel miRNA	193	159	1 943
总数 Total	336	293	3 960

图2 差异表达miRNA靶基因GO富集

Fig. 2 GO enrichment of differentially expressed miRNA targets

图3 差异表达miRNA靶基因KEGG富集

Fig. 3 KEGG enrichment of differentially expressed miRNA targets

图4 差异表达靶基因的启动子顺式作用元件

Fig. 4 Promotor cis-acting elements of differentially expressed targets

图5 抗、感番茄10对miRNA-targets青枯菌侵染下的相对表达量 R和S分别表示抗病和感病番茄。*和**分别表示0.05和0.01水平下的差异显著性，SD表示标准差

Fig. 5 Relative expressions of ten selected miRNA-targets in resistant and susceptible tomato lines with R. solanacearum infection R and S represent resistant and susceptible tomato lines, respectively. * and ** indicate statistically significant differences (P < 0.05 and P < 0.01). SD stands for standard deviation. A: 1: novel_miR_13; 2: Solyc09g091370.4. B:1: sly-miR396a-5p; 2: Solyc02g032870.4; 3: Solyc07g041640.3; 4: Solyc09g009200.3. C: 1: sly-miR172c; 2: Solyc04g064620.4; 3: Solyc10g006710.4; 4: Solyc12g099450.3. D: 1: sly-miR172d; 2: Solyc09g075550.3; 3: Solyc10g006710.4; 4: Solyc12g099450.3

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