本研究以生长在3个不同施氮水平(60kg/hm^2,90kg/hm^2和120kg/hm^)下的“云烟87”为材料,运用超高效液相色谱法、分光光度法和荧光定量PCR法,分别研究了不同施氮水平对烟叶生长发育过程中黄酮类化合物芸香苷、山奈酚-3-O-芸香苷和总黄酮含量、代谢相关酶活性及相关基因表达的影响。结果表明:随着生长发育的进行,黄酮类化合物代谢相关基因PAL、C4H、4CL、CHS、CHI、F3H、F3’H、FLS、A3RT的基因表达总体上呈逐渐降低的趋势,从而调控了山奈酚-3-O-芸香苷和总黄酮含量呈逐渐降低的趋势,芸香苷含量呈先略有升高后降低的趋势。不同施氮水平下比较,处理前期(50d和83d),较低施氮水平(60kg/hm^2)上调了烟草叶片黄酮类化合物代谢相关基因PAL、C4H、4CL、CHS、CHI、F3H、F3’H、FLS、A3RT和ANS的基因表达,提高了黄酮类化合物代谢关键酶PAL、CHI的活性,促进了黄酮类化合物芸香苷、山奈酚-3-O-芸香苷和总黄酮含量的积累0〈0.05)I处理后期(106d),较高施氮水平(120kg/hm^2)上调了烟草叶片黄酮类化合物代谢相关基因PAL、C4H、4CL、CHS、CHI、F3H、F3’H、A3RT和ANS基因的表达,提高了黄酮类化合物代谢关键酶PAL、CHI的活性,促进了黄酮类化合物芸香苷、山奈酚-3-O-芸香苷和总黄酮含量的积累60〈0.05)。
In this study, the tobacco cultivar "Yunyan 87" plants were grown under three different nitrogen levels (60 kg/hm^2, 90 kg/hm^2 and 120 kg/hm^2), and effects of the nitrogen levels on the contents of flavone compounds rutin, kaempferol-3-O-rutin and total flavonoids, the avtivity of metabolism-related enzymes and gene expression in tobacco leaves were investigated during the growth and development of tobacco plants by using ultra perfor- mance liquid chromatography, spectrophotometry and fluorescence quantitative PCR methods. The results showed that, with the growth and development of tobacco leaves, the gene expression of flavonoid metabolism-related genes PAL, C4H, 4CL, CHS, CHI, F3H, F3'H, FLS, A3RTwere gradually decreased in general, which in turn led to the decline trend of the content of Kaempferol-3-O-rutin and total flavonoids, while the content of rutin was first increased slightly and then decreased. Under different nitrogen levels, at the early stage of the treatments (50 days and 83 days), lower nitrogen level (60 kg/hm2) enhanced the gene expression of the flavonoids metabolism-related genes PAL, C4H, 4CL, CHS, CHI, F3H, F3'H, FLS, A3RT and ANS of the tobacco leaves, raised the key enzyme activities of PAL and CHI related to flavonoids metabolism, and finally promoted the accumulation of the flavonoids rutin, kaempferol-3-O-rutin and total flavonoids (p 〈0.05) in tobacco leaves; At the late period of the treatments (106 days), higher nitrogen level (120 kg/hm2) enhanced gene expression of the flavonoids metabolism related genes AL, C4H, 4CL, CHS, CHI, F3H, F3'H, FLS, A3RT and ANS in the tobacco leaves, improved the key enzyme activities of PAL and CHI related to flavonoids metabolism, and finally promoted the accumulation of the flavonoids rutin, kaempferol-3-O-rutin and total flavonoids (P〈0.05) in tobacco leaves.