Research On Comparative Proteomics Of Tobacco Root Tips After Topping

Topping(removal of the flowering head and young leaves)is an essential cultivating measure for flue-cured tobacco which switches the plant from reproductive to vegetative phase.Tobacco has many responses to topping,such as changes in relationship of sink and source,altering distribution of hormone,the second growth of roots,the increase of nicotine biosynthesis in roots,and so on.The industrial usability of upper leaves is reduced by the increase of nicotine accumulated in upper leaves.In order to improve the industrial usability of upper leaves,many measures,such as balanced fertilization and altering the numbers of leaves remained,were performed,but their effects are unsatisfactory.To clarify the response of flue-cured tobacco roots to topping,the suppression subtractive hybridization(SSH)library,differential miRNAs and digital gene expression profile before and after topping were analyzed in our previous work,and some topping responsive genes were identified.Comparative proteomics of tobacco root tips after topping was analyzed in this study,and the main research contents are as follows.1.It is well-known that plant roots contain low protein content and a large number of interfering substances which severely disturb protein extraction and two-dimensional electrophoresis analysis.Therefore,the extraction of high-quality protein from tobacco roots for proteomic analysis is considered as a big challenge.The three modified protein extraction methods(trichloroacetic acid-acetone method,phenol extraction method,trichloroacetic acid-acetone-phenol method)for tobacco roots protein were compared and evaluated through protein yields,sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and two-dimensional electrophoresis.It was proved that the trichloroacetic acid-acetone-phenol protocol is an efficient method for the extraction of tobacco roots proteome.2.The 2-DE map of comparative proteomics of tobacco roots after topping was obtained,and thirteen differential proteins were screened and identified by mass spectrometer.Of these differential proteins,F-box?Ras?LRR?Cal?FPS and bHLH protein are related to biological and abiotic stress response.3.bHLH,MYC1a/b and MYC2a/b belong to bHLH family,and they were proved to be related to the regulation of nicotine biosynthesis.Phylogenetic tree analysis of bHLH transcription factor family in Nicotiana tabacum showed that bHLH and MYC do not belong to the same subfamily.The alignment of tobacco MYC and bHLH proteins indicates that MYC1a/b and MYC2a/b have JAZ binding region and bHLH protein has not JAZ binding region.Therefore,it is possible that bHLH protein is not involved in JA-mediated regulation of nicotine biosynthesis.4.The role of bHLH,MYC1a/b and MYC2a/b in regulating nicotine biosynthesis was analyzed in this study.Since the five bHLH family members have been proved to be involved in regulation of nicotine biosynthesis,their regulatory mechanism is different.It was found that MYC2 a/b is related to nicotine biosynthesis regulated by MeJA and topping,that bHLH protein is related to nicotine biosynthesis induced by drought,salt and cold stress,and that MYC1 a/b is involved in nicotine biosynthesis induced by drought stress.