RNA沉默手段分析烟草悬浮细胞中线粒体分裂蛋白NtFIS1A/1B功能

Functional Validation of Mitochondrial Fission Proteins, NtFIS1A/1B, in Nicotiana tabacum ‘BY-2’ Suspension Cells Using RNA Interference

摘要:

为了解烟草悬浮细胞中的线粒体分裂蛋白的功能,将拟南芥(Arabidopsis thaliana)的线粒体分裂复合体成员FIS1A和FIS1B在烟草(Nicotiana tabacum)表达序列数据库中进行序列比对,鉴定到烟草中的同源基因NtFIS1A 和NtFIS1B (NtFIS1A/1B).以烟草悬浮细胞为材料,通过RNA 干扰和人工microRNA 干扰技术抑制NtFIS1A/1B 的表达, RT-PCR 分析结果表明,RNA 沉默细胞系中NtFIS1A/1B 的转录受到抑制.观察RNA 沉默细胞系中的线粒体形态可见,当NtFIS1A/1B 的表达被抑制后,单个线粒体的平均面积显著增加.这些表明NtFIS1A/1B 参与了烟草悬浮细胞中线粒体分裂的调控,有助于了解烟草中线粒体的形态调控.

Abstract:

The aim was to understand the function of mitochondrial fission proteins. Two homologues, NtFIS1A and NtFIS1B, were identified by searching tobacco (Nicotiana tabacum) EST database with mitochondiral fission member of FIS1A and FIS1B in Arabidopsis. Expression of NtFIS1A/1B in tobacco ‘BY-2’ suspension cells were inhibited by using RNA interference and artificial microRNA interference. The RT-PCR results showed that the transcription of NtFIS1A/1B were knockdown in related RNAi cell lines. Thereafter, mitochondrial morphology in RNA knockdown cell lines was analyzed. The average area of single mitochondria significantly increased. It was concluded that NtFIS1A/1B participated in mitochondrial fission in ‘BY-2’ suspension cells. It would contribute to understand morphological regulation of mitochondria in tobacco suspension cells.