RNA m1A和m5C甲基化修饰在拟轮枝镰孢伏马毒素生物合成中的作用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0213

摘要/Abstract

摘要：

【目的】拟轮枝镰孢（Fusarium verticillioides）是一种危害严重的植物病原真菌，极大降低了粮食产量，还产生2B类致癌物伏马毒素，威胁人畜健康。探究RNA甲基化修饰与伏马毒素之间的联系，解析RNA修饰甲基化转移酶在伏马毒素合成中的作用。【方法】利用HPLC检测了不同地区的拟轮枝镰孢菌株的伏马毒素合成能力，采用QuEChERS前处理结合超高效液相色谱-串联质谱（UPLC-MS/MS）技术建立了m⁶A、m^lA、m⁵C、Gm、m⁷G和Um等6种RNA甲基化修饰检测方法，继而对不同产毒菌株的RNA甲基化修饰进行了检测，并采用生物信息学和RT-qPCR方法确定了与伏马毒素合成相关的RNA甲基化修饰基因。【结果】不同地区的拟轮枝镰孢菌株伏马毒素合成能力具有显著差异，成功建立了RNA甲基化修饰检测方法，并确定m^lA和m⁵C RNA甲基化修饰与伏马毒素合成负相关，RT-qPCR发现Fvalyref基因负调控伏马毒素生物合成。【结论】RNA m⁵C甲基化修饰与伏马毒素生物合成呈负相关且其Reader基因Fvalyref负调控伏马毒素生物合成。

关键词: 拟轮枝镰孢, RNA甲基化修饰, 检测, 伏马毒素

Abstract:

【Objective】Fusarium verticillioides is a serious plant pathogen fungus, which greatly reduces grain yield. It also produces class 2B carcinogen fumonisin, which threatens human and animal health. To explore the relationship between RNA methylation modification and fumonisin, the role of RNA methylated transferase in fumonisin synthesis was analyzed.【Method】 HPLC was applied to determine the fumonisin synthesis ability of F. verticillium strains from different regions. Six kinds of RNA methylation modification detection methods, including m⁶A, m^lA, m⁵C, Gm, m⁷G, and Um, were established by QuEChERS pretreatment combined with ultra-high performance liquid chromatograph-tandem mass spectrometry（UPLC-MS/MS）. Then the RNA methylation modifications of different toxin-producing fungi were detected, and the RNA methylation modification genes related to fumonisin synthesis were identified by bioinformatics and RT-qPCR.【Result】It was found that there were significant differences in fumonisin synthesis ability of F. verticillium strains in different regions, the detection method for RNA methylation modification was successfully constructed, the methylation of RNA m^lA and m⁵C was negatively correlated with fumonisin synthesis, and RT-qPCR showed that Fvalyref gene negatively regulated fumonisin biosynthesis.【Conclusion】The methylation modification of RNA m⁵C and its Reader gene Fvalyref negatively regulate fumonisin biosynthesis.

Key words: Fusarium verticillioides, RNA methylation modification, detection, Fumonisin

侯智涵, 郝楠, 李佳琪, 赵斌, 刘颖超. RNA m¹A和m⁵C甲基化修饰在拟轮枝镰孢伏马毒素生物合成中的作用[J]. 生物技术通报, 2024, 40(9): 282-290.

HOU Zhi-han, HAO Nan, LI Jia-qi, ZHAO Bin, LIU Ying-chao. Roles of RNA m¹A and m⁵C Methylation Modifications in the Fumonisin Biosynthesis of Fusarium verticillioides[J]. Biotechnology Bulletin, 2024, 40(9): 282-290.

图/表 8

表1 试验所用引物

Table 1 Primers used in the experiment

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
18S ribosomal RNA-F 18S ribosomal RNA-R Fvnsun2-F Fvnsun2-R Fvnsun4-F Fvnsun4-R Fvalyref-F Fvalyref-R	GGCCGTTCTTAGTTGGTGGA TGCGGCCCAGAACATCTAAG AGACCTTCCGCAAGCTTCTC CGCTCTCTAAAGTCGGGGTC TGGGTGGTTTCGATCGTGTT TAAACAATGTAGCCGCCCGT AGGCCGTGCTATTGAAGTCC AAAGGGTTAGGAGGGACGGA

表2 不同地区拟轮枝镰孢菌株FB1含量测定

Table 2 Determination of FB1 content of F. verticillioides strain in different regions

菌株编号 Strain number	菌株来源 Strain source	FB₁含量 Content of FB₁/（mg·g^-1）
87	14-SD-94-6	145.20
64	HK12-5	116.90
114	14-17-15	228.20
126	14-SD-91-13	116.80
81	HN 22-2	112.30
104	25-4	89.55
50	LZ-2-81	103.57
80	NMG-1-2	92.44
84	124-3	64.73
86	HN 24-2	99.39
63	S-9-7	101.82
6	M-I1-46	59.29
38	M-10-48	ND
1	LZ-2-77甘	2.18
49	P-HB-T3-4	2.19
55	NMG-3-3	ND
109	M-3-20	ND
36	26-4	ND
18	14-SD-22-20	ND
37 22	14-SD-91-9 M-4-78	ND ND
40 139	ZL-1-6 14-SD-91-9	ND 1.81
33	P-C1-32-1	ND

图1 产毒差异菌株致病性观察

Fig. 1 Observation on the pathogenicity of toxicity-yielding differential strains

表3 6种目标核苷的保留时间和质谱参数

Table 3 Retention time and mass spectrum parameters of 6 target nucleosides

核苷Nucleotide	母离子Precursorion（m/z）	子离子Production（m/z）	保留时间Residence time/ ms	去簇电压DP/V	碰撞电压CE /V
Um	259.3	113.1	0.94	17	14
Gm	298.2	152.1	1.10	8	16
m¹A	282.1	150.1	0.72	36	30
m⁶A	282.2	150.2	2.40	39	20
m⁵C	258.3	126.1	0.68	24	18
m⁷G	298.1	166.1	0.76	20	18

表4 6种核苷的线性方程、决定系数、平均回收率以及相对标准偏差

Table 4 Linear equation, determination coefficient, average recovery, and relative standard deviation of 6 nucleosides

核苷 Nucleotide	检出限LOD /（μg·kg^-1）	基质效应 Matrix effect/%	线性方程 Linearity equation	决定系数R² Determination coefficient	平均回收率/相对标准偏差 Average recovery（%）/ RSD
核苷 Nucleotide	检出限LOD /（μg·kg^-1）	基质效应 Matrix effect/%	线性方程 Linearity equation	决定系数R² Determination coefficient	1 μg/kg	20 μg/kg	500 μg/kg
Um	0.002	5.41	y =4E+06x - 2964.3	0.9979	91.9/4.7	84.4/4.5	91.9/3
Gm	0.001	2.27	y =2E+06x - 5329.46	0.9999	97.6/6.6	86.2/7.8	109.7/4.9
m¹A	0.0009	2.16	y = 7E+07x - 161526	0.9990	72.5/4.9	75.1/1.9	74.4/7.6
m⁶A	0.0006	2.13	y = 7E+07x - 30294	0.9981	88.5/7.0	78.7/5.9	116.5/1.4
m⁵C	0.0003	2.51	y = 3E+06x - 1896.4	0.9993	70.3/6.5	71.0/4.7	74.1/7.2
m⁷G	0.0006	1.95	y = 6E+07x - 141064	0.9995	74.0/8.9	72.1/7.7	70.9/9.5

图2 产毒差异菌株中RNA修饰的变化 A：Um修饰含量；B：Gm修饰含量；C：m6A修饰含量；D：m7G修饰含量；E：m5C修饰含量；F：m1A修饰含量。High-toxicity-producing strains缩写为High-；Non-toxicity-producing strains缩写为Non-。图中数据为平均值±标准误差，ns：无显著性差异，*（P<0.05），**（P<0.01），***（P<0.001）。下同

Fig. 2 Changes of RNA modification in toxicity-yielding differential strains A: Um modification content; B: Gm modification content; C: m6A modification content; D: m7G modification content; E: m5C modification content; F: m1A modification content. High-toxicity-producing strains abbreviated as High-. Non-toxicity-producing strains abbreviated as Non-. Data in the figure are mean ± SE. ns: No significant difference，* (P<0.05), ** (P<0.05), *** (P<0.001). The same below

图3 m5C修饰相关基因生物信息学分析

Fig. 3 Bioinformatics analysis of m5C modification-related genes

图4 m5C修饰相关基因在不同产毒菌株中的表达 A：Fvalyref在不同含量伏马毒素菌株表达量；B-C：Fvnsun2与Fvnsun4在不同含量伏马毒素菌株表达量

Fig. 4 Expression of m5C modification-related genes in different toxin-producing strains A: Expression of Fvalyref in the strains with different fumonisin contents; B-C: Expressions of Fvnsun2 and Fvnsun4 in the strains with different fumonisin content

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RNA m¹A和m⁵C甲基化修饰在拟轮枝镰孢伏马毒素生物合成中的作用

Roles of RNA m¹A and m⁵C Methylation Modifications in the Fumonisin Biosynthesis of Fusarium verticillioides

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