转录后基因沉默系统研究烟草rbcS基因功能

The Analysis of rbcS Gene Function by Post-transcription Gene Silencing in Nicotiana benthamiana

初步建立了利用病毒载体诱导转录后基因沉默系统研究烟草(Nicotianabenthamiana)1,5 二磷酸核酮糖羧化酶/加氧酶小亚基(Ribulose 1,5 bisphosphatecarboxylase/oxylasesmallsubunit,rbcS)基因功能的模式。用携带与1,5 二磷酸核酮糖羧化酶/加氧酶小亚基基因同源的cDNA片段的烟草脆裂病毒载体(pTV.rbcS)侵染烟草(Nic otianabenthamiana),诱导内源rbcS基因沉默并在此基础上建立了研究rbcS基因功能的模式:初步进行了rbcS基因沉默后的表型分析、转录水平分析、蛋白质表达水平分析以及利用HPLC方法定量分析rbcS基因沉默后的光合色素变化。结果表明:病毒诱导基因沉默瞬时表达体系中烟草最佳侵染时期为苗龄21～24d,用于侵染的重组农杆菌的最佳浓度的OD值为1～1.5;烟草Rubisco小亚基的表达量可能调节Rubisco大亚基的表达量;烟草rbcS基因与光合作用中的光能收集无关。对rbcS基因沉默的烟草叶片及对照烟草叶片的部分重要光合作用指标分析表明, 运用烟草脆裂病毒载体诱导转录后基因沉默系统研究烟草rbcS基因功能具有可行性,为进一步深入研究rbcS基因功能奠定了基础。

A system of virus induced post transcriptional gene silencing for studying rbcS gene function was been established and optimized using tobacco rattle virus vector and Nicotiana benthamiana as experimental materiaes.The following analyses were conducted:phenotypic characterigation of rbcS gene silenced plants,transcription levels of rbcS gene by RT PCR;protein levels of rbcS by the antibodies of rbcS and rbcL and photosynthetic pigments wntents in rbcS silenced plants by HPLC method.The results showed that the seedlings at 21 24 day old and Agrobacterium concentration at OD 600 =1 1 5 gave the best results for gene silencing.The expression level of rbcL was very likely regulated by rbcS ,and rbcS gene did not relate to the collection of photosynthetic energy.Probability analysis showed that the tobacco rattle virus vector system is a useful and effective technique to study rbcS gene function via post transcriptional gene silencing.