| The nicotine is a tobacco-specific alkaloid, a secondary metabolitesynthesized in the root. After topping，the content of nicotine accumulated, but thereason why the content increased and the regulation mechanism of nicotine are notclear. It is reported that the proper reason of nicotine biosynthesis strengthen aftertopping is the changing of plant hormone source and injure stimulus or secondarydevelopment of the root. In order to research the regulatory mechanism of nicotinebiosynthesis enhanced afer topping, In this experiment，we use tobacco K326cultivated on field and in laboratory as experimental materials,the experiment choseplant grew uniform and carried out four processing methods include un-topping,topping, spraying MeJA without topping and spraying NAA with topping. Weanalyzed the effect on nicotine biosynthesis from the changing of hormone source,the development of root system and the injure stimulus signal MeJA.The mainlyresult is as follows:（1）Real-time PCR technique was applied to verify miRNA expresseddifferently in high flux sequencing database of miRNA before and after topping. Itshowed that miRNA399c, miRNA171, miRNA101d, miRNA166c, miRNA167d hadan increased expression level at24h after topping while miRNA164, miRNA166,miRNA169c, miRNA156, miRNA159had an decreased expression level at24h aftertopping.（2）RT-PCR and Northern blot was applied to check expressing of geneNtNAC-R1and putrescine N-methyl transferase (PMT)in organization of root tips ofthe plants had been treated by the four methods, and discovered that the expressionlevel of NtNAC-R1and PMT increased notably after topping; the expression ofPMT increased while it declined with NtNAC-R1after spraying MeJA onun-topping; the expression of NtNAC-R1increased while it is not significant changewith PMT after topping with spraying NAA. Checked the expression of NtNAC-R1gene in the organization of root tips of the plants without topping and that withtopping2,4,6,8,10,24hours，it showed: the expression of NtNAC-R1increasedfirstly then declined in4hours after topping.（3）Used ELISA to check content of IAA in organization of root tips of theplants had been treated by the four methods, the results were that the content of IAA declined obviously in root with top pruning and spraying NAA on topping, yet,that kept still with spraying MeJA on un-topping.（4） Phase chromatography was applied to check nicotine content inorganization of root tips of the plants had been treated by the four methods,the resultproved the content of nicotine rose somewhat in root tip of plant with top pruningand spraying MeJA on un-top pruning, yet that reduce a little with spraying NAA ontop pruned ones.（5）Used Root scanner to check phenotype of root system of the tobacco hadbeen treated, it turned out: root system grew flourishing obviously, laterals increasedand dry matter accumulated rise after topping, yet it has not changed much withplant which had sprayed MeJA on un-topping plant and had sprayed NAA ontopping.All the results above indicate topping had prompted the development of rootsystem and the increasing of branch root surely, it maybe the result tempted bychanging of auxin IAA, otherwise the changing of increased expression of PTMtempted by topping had clear relationship with hormone source. But there had norelationship between increased expression of PTM tempted by MeJA andsecondary development of root system.We surmised there were two reasons forincreased synthesis ability of nicotine in the root tempted by topping: for one, thesecondary development of root system had increased its volume and the place whichsynthesis nicotine; for the other, topping had tempted the increasing of injurestimulus signal MeJA and the IAA source reducing, which tempted increasingexpression of PMT and the synthesis ability of nicotine. therefore, changing ofhormone source, the secondary development of root system and injure stimulussignal MeJA working together leaded the increasing of nicotine biosynthetic aftertop puring in tobacco. |
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