基于转录组分析的SlCMT4基因敲除对番茄花器官的影响

Effects of SlCMT 4 gene knockout on tomato floral organs based on transcriptome analysis

[目的]DNA甲基化是由DNA甲基转移酶催化的重要表观遗传修饰,DNA甲基转移酶在植物发育、转录控制和代谢途径调控中具有重要作用。本研究旨在明确DNA甲基转移酶基因SlCMT4调控番茄花器官形成和发育的表观遗传机制。[方法]本研究以经典番茄栽培品种Ailsa Craig(AC^(^(++)))为背景材料,采用CRISPR⁃Cas9基因编辑技术,获得SlCMT4突变株系。我们提取番茄花器官RNA,进行转录组测序,根据差异基因进行基因功能及代谢通路分析。[结果]研究了SlCMT4基因在野生型(AC^(++))和突变体番茄(Nr和Rin)不同果实发育阶段的表达模式,发现该基因在番茄成熟突变体Nr和Rin果实中的表达水平显著高于野生型番茄,表明SlCMT4基因的表达可能受激素乙烯信号和成熟相关转录因子RIN的负调控。SlCMT4基因敲除导致番茄花器官出现多种表型,包括雄蕊卷曲、雌蕊变粗变短等。我们在突变株系(CR⁃08)的花器官样品中鉴定了1398个差异表达基因(DEG),其中512个上调,886个下调。差异表达基因的KEGG分析结果表明,大多数差异表达基因被归类为以下5条功能代谢通路:植物激素信号转导、苯丙烷生物合成、淀粉和蔗糖代谢、氨基糖和核苷酸糖代谢。参与生长素激活信号通路与木葡聚糖代谢过程的基因下调表达较多,乙烯生物合成与细胞分裂素代谢过程的基因上调表达较多,这些代谢途径可能在番茄花器官形成和发育中发挥了重要的作用。[结论]从转录组水平研究了DNA甲基转移酶基因SlCMT4基因的功能,研究结果表明SlCMT4基因可能在调控番茄花器官形成和发育中发挥重要作用,为进一步探索番茄DNA甲基转移酶基因的功能提供新的理论基础。

[Objective]DNA methylation is an important epigenetic modification catalyzed by DNA methyltransferase,which plays a crucial role in plant development,transcriptional control,and metabolic pathway regulation.This study aimed to deter-mine the epigenetic mechanisms through which the DNA methyltransferase gene SlCMT4 regulates the growth and develop-ment of tomato floral organs.[Methods]In this study,the classic tomato cultivar Ailsa Craig(AC^(++))was used as the back-ground material to obtain SlCMT4 mutant lines by employing CRISPR/Cas9 gene editing technology.We extracted RNA from tomato floral organs,performed transcriptome sequencing,and analyzed gene function and metabolic pathway based on differentially expressed genes.[Results]We studied the expression patterns of the SlCMT4 gene at different fruit development stages of the wild type(AC^(++))and mutant tomatoes(Nr and Rin).It was found that the gene’s expression in the mature mu-tant tomatoes Nr and Rin fruits was significantly higher than in wild type tomatoes,suggesting that the expression of the SlC-MT4 gene may be negatively regulated by the hormone ethylene signal and the maturation-related transcription factor RIN.Knocking out the SlCMT4 gene resulted in a various phenotypic changes in tomato floral organs,including curled stamen and thickened and shortened pistils.We identified 1398 differentially expressed genes(DEGs)in the floral organ samples of the mu-tant line CR-08,with 512 up-regulated and 886 down-regulated genes.KEGG analysis of the differentially expressed genes showed that most of them were grouped into the following five functional metabolic pathways:plant hormone signal transduc-tion,phenylpropanoid biosynthesis,starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism.Genes in-volved in the auxin activation signal pathway and cellulose metabolism were predominantly down-regulated,while those related to ethylene biosynthesis and cytokinin metabolism were mostly up-regulated.These metabolic pathways likely play important roles in the formation and development of tomato floral organs.[Conclusion]We conducted a functional study of the DNA meth-yltransferase gene SlCMT4 at the transcriptomic levels.The results suggested that SlCMT4 gene may play an important role in regulating the formation and development of tomato floral organs,providing a new theoretical foundation for further exploration of the functions of tomato DNA methyltransferase gene.