基于转录组和RT-qPCR技术挖掘烟草果胶水解酶基因
Mining of tobacco pectin hydrolase genes based on transcriptomics and RT-qPCR
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摘要: 【目的】 筛选产果胶酶菌株,挖掘烟草果胶降解关键酶基因。【方法】 采用3,5-二硝基水杨酸比色(DNS)法,以烟草果胶为唯一碳源筛选烟草果胶降解菌株,利用转录组技术分析烟草果胶降解过程中相关酶的差异表达,通过实时荧光定量PCR(RT-qPCR)技术验证并确定烟草果胶降解关键酶基因。【结果】 筛选得到一株烟草果胶降解菌株GB3,在发酵液降解烟草果胶中酶活力值为32.95 U/mL,通过形态学和16S rDNA序列分析,初步鉴定为变栖克雷伯杆菌(Klebsiella variicola);GB3在降解普通果胶过程中有1076个基因上调,而降解烟草果胶过程中有1100个基因上调;与烟草果胶降解相关差异表达基因主要富集于半乳糖代谢、糖酵解/糖异生和其他聚糖降解途径,共筛选到9个可能与烟草果胶降解相关的酶基因,经RT-qPCR分析,rlpA、lacZ、ogl、rhaA和P48843为烟草果胶降解关键酶基因。【结论】 验证了烟草果胶降解关键酶基因为gene1209,gene1526,gene2083,gene310和gene4941,为研究果胶酶基因,降低烟叶果胶含量,提升烟叶品质提供参考。Abstract: 【Objective】 This study aimed to screen pectinase-producing strains and mine the key genes involved in tobacco pectin degradation. 【Methods】 Pectinase-producing strains were screened using the 3,5-dinitrosalicylic acid (DNS) method with tobacco pectin as the sole carbon source. Transcriptomic analysis was performed to examine differential gene expression during tobacco pectin degradation, and key genes were verified and identified by real-time quantitative PCR (RT-qPCR). 【Results】 A tobacco pectin-degrading strain designated GB3 was obtained, and its enzyme activity against tobacco pectin in the fermentation broth reached 32.95 U/mL. The strain was preliminarily identified as Klebsiella variicola based on morphological characteristics and 16S rDNA sequence analysis. During the degradation of common pectin, 1076 genes were upregulated in strain GB3, whereas 1100 genes were upregulated during tobacco pectin degradation. Differentially expressed genes (DEGs) related to tobacco pectin degradation were mainly enriched in galactose metabolism, glycolysis/gluconeogenesis, and other glycan degradation pathways. A total of nine candidate enzyme genes potentially associated with tobacco pectin degradation were screened. RT-qPCR analysis further revealed that rlpA, lacZ, ogl, rhaA, and P48843 were the key genes involved in tobacco pectin degradation. 【Conclusion】 This study confirmed that gene1209, gene1526, gene310, and gene4941 are the key tobacco pectin hydrolase genes, providing a reference for investigating pectinase genes, regulating pectin content in tobacco leaves, and improving tobacco leaf quality.
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Key words:
- tobacco pectin /
- DEGs /
- tobacco pectin hydrolase /
- transcriptome /
- RT-qPCR
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