烟草NtCycB2启动子的克隆和组织驱动特性分析

Molecular cloning and tissue expression analysis of NtCycB2 promoter in tobacco

【背景和目的】烟草B型细胞周期蛋白CycB2可以负调控腺毛的发生,为分析烟草Nt CycB2启动子的组织驱动特性。【方法】通过实时定量PCR分析普通烟草NtCycB2-1和Nt CycB2-2两种序列的组织表达差异,克隆NtCycB2-1和NtCycB2-2启动子序列,对其进行进化分析、顺式元件预测和组织驱动特性分析。【结果】(1)烟草NtCycB2-1和NtCycB2-2均为腺毛优势表达基因,NtCycB2-1的表达量显著高于NtCycB2-2;(2)普通烟草中存在两种NtCycB2启动子序列类型(ProNtCycB2-1和ProNtCycB2-2),其中ProNtCycB2-1来自于林烟草,ProNtCycB2-2来自于绒毛烟草,这些CycB2启动子中普遍存在ABRE等激素应答元件,以及MYB-motif、MYC-motif和TC-rich repeats等逆境响应元件;(3)以GUS为报告基因,研究NtCycB2-1和NtCycB2-2两个启动子的组织驱动特性发现,NtCycB2-1和NtCycB2-2启动子均能驱动GUS基因在叶片和茎部腺毛中特异表达,其中NtCycB2-1启动子的驱动能力高于NtCycB2-2。【结论】Nt CycB2启动子可驱动下游基因在腺毛中特异表达。本研究为寻找腺毛特异表达顺式元件、构建腺毛人工启动子、研究腺毛发生机制奠定基础。

Glandular trichomes are small protrusions of epidermal cells on plant surface.As important secretory tissues in plants,glandular trichomes can specially synthesize a variety of secondary metabolites.These metabolites not only play a pivotal role in plant adaptation to biotic and abiotic stresses,but also have important economic values.The B-type cyclin regulates the transition of G2-to-M in plants.It has been reported that CycB2 negatively regulates the initiation of glandular trichome.In this study,we analyzed the tissue expression patterns of NtCycB2-1 and NtCycB2-2 sequences in common tobacco,cloned the promoter sequences of NtCyCB2-1 and NtCycB2-2,and analyzed their evolutionary relationships,cis-elements and tissue-driven characteristics.The results showed that NtCycB2-1 and NtCycB2-2 were both trichome dominant expression genes,and the expression level of NtCycB2-1 was significantly higher than that of NtCycB2-2.Sequence analysis showed that there were two NtCycB2 promoter sequences(ProNtCYCB2-1 and ProNtCYCB2-2)in Nicotiana tabacum,among which ProNtCYCB2-1 derived from Nicotiana sylvestris and ProNtCYCB2-2 was from Nicotiana tomentosiformis.These tobacco CycB2 promoters contain some hormone and stress responsive elements,such as ABRE,MYB-motif,MYC-motif and TC-rich repeats.Using GUS as the reporter gene,the tissue driving abilities of ProNtCYCB2-1 and ProNtCYCB2-2 were studied in tobacco.It was found that both ProNtCYCB2-1 and ProNtCYCB2-2 can drive GUS gene that is specifically expressed in glandular trichomes of leaves and stems.Notably,ProNtCYCB2-1 promoter had higher driving ability than ProNtCYCB2-2.This study laid a foundation for detecting glandular-trichome specific cis-elements,constructing glandular trichome-specific synthetic promoters,and exploring potential mechanism of the development of glandular trichomes.