烤烟烟碱含量标记基因NtMPO1和NtARF6的鉴定

Identification of Nicotine Content Marker Genes NtMPO1 and NtARF6

为了鉴定有效的烟碱含量标记基因,测定89个烤烟品种叶片烟碱含量,筛选出高、中、低烟碱烤烟品种,然后通过实时荧光定量PCR(qRT-PCR)测定高、中、低烟碱烤烟品种中烟碱合成、转运和调控相关的共25个基因的相对表达量,分析基因表达量与烟碱含量的相关性,筛选烟碱含量候选标记基因,后续以云烟87和K326为材料,通过设置不同氮素浓度(2.5、5.0、7.5 mmol/L NaNO_(3))、采取不同部位(上、中、下部)叶片和打顶前后10 d叶片创制烟碱含量差异材料,测定烟碱含量及其候选标记基因的相对表达量,验证烟碱含量候选标记基因的有效性。结果表明,从89个烤烟品种中筛选出高、中、低烟碱烤烟品种各4个,这些烤烟品种的烟碱含量与NtMPO1基因相对表达量呈极显著负相关,与NtARF6基因相对表达量呈极显著正相关,两者可以作为烟碱含量标记基因的候选基因。烟碱含量候选标记基因的验证试验结果表明,与正常供氮处理相比,高氮处理叶片烟碱含量显著增加,NtMPO1基因相对表达量显著降低,NtARF6基因相对表达量显著提高;低氮处理烟碱含量显著降低,NtMPO1基因相对表达量显著提高,NtARF6基因相对表达量显著降低。不同部位烟叶中,上部叶烟碱含量显著高于中部叶,中部叶烟碱含量显著高于下部叶;上部叶中NtMPO1基因相对表达量显著低于中部叶,中部叶中NtMPO1基因相对表达量显著低于下部叶;上部叶中NtARF6基因相对表达量显著高于中部叶,中部叶中NtARF6基因相对表达量显著高于下部叶。相较于打顶前,打顶后叶片烟碱含量显著提高,NtMPO1基因相对表达量显著降低,NtARF6基因相对表达量显著提高。综上,NtMPO1和NtARF6基因可以作为烟碱含量的有效标记基因。

In order to identify the effective nicotine content marker genes,the nicotine contents of 89 flue⁃cured tobacco varieties were determined,the high⁃,medium⁃and low⁃nicotine flue⁃cured tobacco varieties were screened,the relative expression levels of a total of 25 genes related with nicotine synthesis,transport,regulation in high⁃,medium⁃and low⁃nicotine flue⁃cured tobacco varieties were determined by real⁃time quantitative PCR(qRT⁃PCR),the correlation between relative expression levels of the above genes and nicotine content was analyzed,and candidate marker genes for nicotine content were screened.The leaves of tobacco treated with 2.5、5.0、7.5 mmol/L NaNO_(3),upper,middle and lower parts of leaves and the leaves before and after topping(10 days)were used to determine the nicotine content and the relative expression levels of candidate marker genes,so as to verify the effectiveness of nicotine content candidate marker genes.The results showed that 4 high⁃,4 medium⁃and 4 low⁃nicotine flue⁃cured tobacco varieties were screened from 89 flue⁃cured tobacco varieties,and the nicotine content of these flue⁃cured tobacco varieties was significantly negatively correlated with the relative expression levels of NtMPO1 gene and significantly positively correlated with the relative expression levels of NtARF6 gene,which could be used as candidate genes for nicotine content marker genes.The results of the validation test of nicotine content candidate marker genes showed that compared with the normal nitrogen treatment,the nicotine content of leaves in the high nitrogen treatment significantly increased,the relative expression level of NtMPO1 gene significantly decreased,and the relative expression level of NtARF6 gene significantly increased;the nicotine content of the low nitrogen treatment significantly decreased,the relative expression level of NtMPO1 gene significantly increased,and the relative expression level of NtARF6 gene significantly decreased.The nicotine content of the upper leaves was significantly higher than that of the middle leaves,and the nicotine content of the middle leaves was significantly higher than that of the lower leaves;the relative expression level of NtMPO1 gene in the upper leaves was significantly lower than that in the middle leaves,and the relative expression level of NtMPO1 gene in the middle leaves was significantly lower than that in the lower leaves;the relative expression level of NtARF6 gene in the upper leaves was significantly higher than that in the middle leaves,and the relative expression level of NtARF6 gene in the middle leaves was significantly higher than that in the lower leaves.Compared with before topping,the nicotine content of leaves significantly increased after topping,the relative expression level of NtMPO1 gene significantly decreased,and the relative expression level of NtARF6 gene significantly increased.In conclusion,the NtMPO1 and NtARF6 genes can be used as effective markers of nicotine content.