超量表达AtBAS1基因对烟草中烟碱和降烟碱合成的影响

The Impact of Over-Expression of AtBAS1 Gene on the Synthesis of Nicotine and Nornicotine in Tobacco

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 define the impact of phyB activation-tagged suppressor 1, BAS 1 gene of A rob idopsis tha!iana on the synthesis of tobacco nicotine, nornicotine, and nitroso nornicotine, this study constructed plant expression vector containing different promoters pSH-pLXM5-BA S1 and pSH-35S-BA S1 and obtained the transgenic tobacco plants. Through the same part of the transgenic and wild tobacco leaves during 8-10 leaf stage, the method of LC-MS was adopted to determinate the content of nicotine, nicotine, nornicotine, and nitroso nornicotine on thesample. The results showed that the content of nicotine and nomicotine in transgenic tobacco were significantly higher than those in wildtype. The content of nicotine in pLXM5-BA S1 gene plant and 35S-BA S1 gene plant were 2.9 times and 2.95 times of the wildtype, respectively; at the same time, nomicotine contents were respectively 3.35 times and 3.76 times of the wildtype. Nitroso nomicotine was not detected in the transgenic and wildtype, which meant over-expression of A tBASI had an apparent effect on the synthesis of nicotine and nomicotine in tobacco. According to sequence of PMT, OPT genes related with nicotine and CYP82E4v1, CYP82E5v2 and CYP82E10 in nornicotine synthesis reported in the NCBI database, we designed Real-time PCR primers for each gene and took tobacco fl-A ctin gene as the intemal reference. The results of the Real-time PCR indicated that the expression of nicotine and nicotine synthesis related genes were up-regulated in different degrees. In conclusion, the overexpression of A tBA $1 gene in tobacco caused the up regulation of nicotine and nicotine synthesis related genes, which directly led to a significant increase of nicotine and nicotine content in tobacco, the genetic transformation A tBA S1 gene to tobacco, caused the related gene expression raising of nicotine and nornicotine and directly led to greatly increasing nicotine and nornicotine in tobacco. This study might provide a reference for further research on new genes that affect nicotine and nicotine production and transformation, and finally construct a complete network of nicotine metabolism. Also, it might provide a new way to study the signal factors and signal transduction during the transformation of nicotine into nicotine.