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为明确拟南芥(Arabidopsis thaliana)光敏色素B激活标签的抑制蛋白1(phy B activation-tagged suppressor1,BAS1)基因对烟草(Nicotiana tabacum)中烟碱、降烟碱和N-亚硝基降烟碱合成的影响,通过构建含有不同启动子的植物表达载体p SH-p LXM5-BAS1和p SH-35S-BAS1遗传转化烟草。在8~10叶期分别取转基因和野生型烟草植株相同部位的叶片,采用LC-MS方法对样品中烟碱、降烟碱、亚硝基降烟碱含量进行测定,结果表明转基因烟草烟碱含量和降烟碱含量与野生型相比均明显提高。转p LXM5-BAS1基因和转35S-BAS1基因植株烟碱含量分别是野生型植株2.9倍和2.95倍;同时测得降烟碱含量分别为野生型的3.35倍和3.76倍;在转基因和野生型烟草中均未检测到亚硝基降烟碱。表明超量表达At BAS1对烟草的烟碱和降烟碱合成存在显著影响。据NCBI数据库中报道的烟碱合成相关基因PMT和QPT,降烟碱合成相关基因CYP82E4v1、CYP82E5v2、CYP82E10的序列,设计各基因的Real-time PCR引物,并以烟草β-Actin基因作为内参,Real-Time PCR结果表明烟碱和降烟碱合成相关基因表达均出现不同程度的上调。综上可知,在烟草中超量表达At BAS1基因,促使烟碱和降烟碱合成相关基因表达的上调,直接导致了烟草中烟碱含量和降烟碱含量的大幅提高。本研究为后续深入挖掘影响烟碱及降烟碱生成及转化的新基因并最终构建完整的烟碱代谢网络提供了借鉴,也为研究烟碱向降烟碱转化过程中信号因子和信号传递提供了一个新途径。
In order to clarify the inhibitory effects of phy B activation-tagged suppressor1 (BAS1) gene of Arabidopsis thaliana on nicotine, nornicotine and N-nitroso-reductase in tobacco (Nicotiana tabacum) Alkali-synthesis, tobacco was genetically transformed by constructing plant expression vectors p SH-p LXM5-BAS1 and p SH-35S-BAS1 containing different promoters. The contents of nicotine, nornicotine and nitroso-nornicotine in the samples were determined by LC-MS at 8-10 leaf stage respectively. The results showed that nicotine, Content and nornicotine content were significantly increased compared with the wild type. The nicotine contents in transgenic plants pLXM5-BAS1 and transgenic 35S-BAS1 were 2.9 times and 2.95 times higher than those in wild type plants, respectively. The nornicotine concentrations were 3.35 and 3.76 times of that in wild type plants, respectively. Nitroso-nornicotine was not detected in tobacco. This indicated that overexpression of At BAS1 had a significant effect on nicotine and nornicotine synthesis in tobacco. Real-time PCR primers of each gene were designed according to the sequences of the genes related to nicotine synthesis, PMT and QPT reported in the NCBI database, and the genes related to nornicotine synthesis. CYP82E4v1, CYP82E5v2, The results of -Time PCR showed that nicotine and nornicotine-related genes were up-regulated to some extent. In conclusion, the overexpression of At BAS1 gene in tobacco promotes the up-regulation of nicotine and nornicotine-related gene expression, which directly leads to the substantial increase of nicotine and nornicotine in tobacco. This study provides a reference for further exploration of new genes that affect the formation and transformation of nicotine and nornicotine, and ultimately construct a complete network of nicotine metabolism, as well as for the study of signal factors and signaling during nicotine conversion to nornicotine A new way.