NtMYB4a基因对烟草多酚类物质合成的影响  

Effect of NtMYB4a Gene on Synthesis of Tobacco Polyphenols

作　　者：杨琴 罗倩 孙光军 姜超英 聂琼[1,2] Yang Qin;Luo Qian;Sun Guangjun;Jiang Chaoying;Nie Qiong(College of Agriculture,Guizhou University,Guiyang,550025;Guizhou Key Laboratory of Tobacco Quality Research,Guizhou University,Guiyang,550025;Guizhou Tobacco Company,Guiyang,550081)

机构地区：[1]贵州大学农学院,贵阳550025 [2]贵州大学,贵州省烟草品质研究重点实验室,贵阳550025 [3]贵州省烟草公司,贵阳550081

出　　处：《分子植物育种》2021年第8期2588-2595,共8页Molecular Plant Breeding

基　　金：贵州省科技计划项目([2019]1409);贵州大学人才引进项目(2018038)共同资助。

摘　　要：为了解NtMYB4a基因的表达是否与烟草多酚化合物的合成有关,本研究以NtMYB4a过表达和CRISPR/Cas9敲除植株及其野生型植株为试验材料,采用实时荧光定量PCR技术分析NtMYB4a和多酚物质合成相关酶基因PAL、C4H、4CL、HCT的表达,使用HPLC方法测定烟叶中绿原酸、芦丁等8种多酚物质的含量,并进行相关性分析。结果表明,过表达NtMYB4a植株中,除花中的C4H外,茎、叶、花中PAL、C4H、4CL和HCT的表达量均极显著高于野生型,烟株茎、叶、花中绿原酸含量也显著增加;敲除NtMYB4a使烟株花、茎和叶中PAL、C4H、4CL和HCT的表达均下调,其中茎中PAL、HCT的表达极显著下调,使叶和花中的绿原酸、芦丁、槲皮素、莨菪亭含量均显著降低,茎中芦丁含量则显著增加,而山奈酚、芥子酸、对香豆酸和咖啡酸含量无显著性变化;NtMYB4a的表达量与绿原酸含量呈极显著正相关关系,与芦丁、莨菪亭、山奈酚、芥子酸、槲皮素、对香豆酸、咖啡酸含量无显著相关性。因此,NtMYB4基因对烟草绿原酸合成相关酶基因表达及绿原酸合成具有调控作用。本研究结果为进一步阐明烟叶中绿原酸形成的分子机制提供理论依据。In order to explore whether the NtMYB4 a gene is involved in the synthesis of polyphenol compounds in tobacco, NtMYB4 a overexpression and knockout plants and their wild-type plants were as materials. The expression of NtMYB4 a and polyphenol synthesis-related enzyme genes PAL, C4 H, 4 CL and HCT were analyzed by real-time fluorescent quantitative PCR. The content of 8 kinds of polyphenols in tobacco were determine using HPLC, and the correlation between the expression of NtMYB4 a gene and the content of polyphenols was analyzed.The results showed that overexpression of NtMYB4 a can significantly up-regulate the expression of PAL, C4 H,4 CL and HCT in flowers, stems, and leaves of tobacco plants, except for C4 H in flowers, which resulted in significantly increase the content of chlorogenic acid in tobacco plants’ stems, leaves and flowers. However, CRISPR/Cas9-mediated knockout of NtMYB4 a downregulated PAL, C4 H, 4 CL and HCT, among them PAL and HCT in the stems were significantly down-regulated, which resulted in reduced the content of chlorogen acid, rutin, quercetin,and scopoletin in plant leaves and flowers, while the content of rutin in the stem was significantly increased.Overexpression and knockout of NtMYB4 a were no significant effect in the content of rutin, scopoletin, kaempferol,erucic acid, quercetin, p-coumaric acid, and caffeic acid. The expression level of NtMYB4 a was significantly positively correlated with the content of chlorogenic acid. Therefore, the NtMYB4 a gene has potent regulatory control on chlorogenic acid synthesis-related gene expression and promotes the synthesis of chlorogenic acid. The results provide a theoretical basis for further elucidating the molecular mechanism of chlorogenic acid formation in tobacco leaves.

关 键 词：烟草 NtMYB4a 基因表达 多酚 相关性分析 

分 类 号：S572[农业科学—烟草工业]