Study On Molecular Regulation Mechanism Of The Nornicotine Biosynthesis In Nicotiana Tabacum L.

Nicotine is the major alkaloid of Nicotiana tabacum L,accounting for 90%-95%,while the nornicotine below 5% normally.The nicotine has significant effect on the quality of tobacco,but the nicotine is mostly transformed into nornicotine during the maturity,senility and baking processes of tobacco,which makes nornicotine to reach the highest amount of alkaloid.The nornicotine can affects the quality of tobacco,and serve as synthesis of precursor for the nitrosonornicotine,which has the potential to cause cancer and harmful to human health.So how to effectively manipulate the nicotine at appropriate level,and reduce the nornicotine content as far as possible are the main points of the tobacco breeding research.By using the genetic engineering technology,our research reveal the molecular mechanism of metabolism regulation of nornicotine and the regulating method of reducing the nornicotine at molecular level,which is helpful to the basis of tobacco breeding.The following results were obtained:(1)The content and gene expression analysis of nicotine and nornicotine for Different types of tobacco germplasmThe nicotine,nornicotine and nitrosonornicotine amount of 7 tobacco species were measured by the UPLC-MS/MS technology.The results showed nicotine were ranked from high to low as N.rustica,burley tobacco,flue-cured tobacco,N.glutinosa,sun-cured tobacco,aromatic tobacco,air-cured tobacco;Nornicotine were ranked from high to low as flue-cured tobacco,burley tobacco,N.rustica,N.glutinosa,sun-cured tobacco,aromatic tobacco,air-cured tobacco.The results of Real-Time PCR analysis indicated thatthe expression level of all genes in N.rustica was significant highest others.The expressing levels of other genes varied greatly,of which the PMT and A622 shared similar expressing mode,while expressing level of nicotine demethylase in different tobacco species differentiate greatly.In N.rustica,the expression of CYP82E4v1 gene increased to 154.7 times higher comparing with K326,while the TN90 was lowest(5.8% of K326).On the contrary,the CYP82E5v2 owned relatively higher expression level(1.98 times of K326)in Florida301 and lowest in N.glutinosa(20.78% of K326).Besides,the expression of CYP82E10 and CYP84E4v1 shared the same expressing situation,and 10.53 times higher in N.rustica than K326.The results demonstrated the great different content of the two alkaloid in various tobacco,which was the results of synergistic effect of related genes through the metabolism pathway.(2)The effects of PMT and QPT on the biosynthesis of nicotineIn this study,by the agrobacterium-mediated method,the plant expression vector that over express or interferingly express of PMT,QPT were transfected in the tobacco species.After the GUS histochemical stain and PCR examination,a total of 33 strains were acquired.The results of expression measurement indicated that the nicotine content of the over expressing PMT was increasing 93.2% compared with wild type.And the average nornicotine content was 2.2 times higher compared with wild type.The over expressing QPT strains no significant difference compared with wild type.But the transgenetic PMT and QPT tobacco strains decreasing 31.5% and 60% respectively compared with wild type,the maximum decreasing extent was 75.9% strikingly.These results proved the over expression of PMT would increase the content of the nicotine and nornicotine significantly.The related gene expression analysis showed,the expression of PMT in the over-expression PMT strains was 20 times higher compared with wild type,while decrease 26.3% in the over-expression QPT strains,which testified the interfering vector inhibited the expression of PMT.The expression of QPT in over-expression QPT strains was higher than the control with the maximum increment of 46.2%;the similar results was shared by the PMT expression with the maximum increment of 69.7%;beyond our anticipation,in the interfering-expression strains,the expression of QPT was not inhibited.The expression of A622 in the QPT transgenetic strain decreased to 0.8%—37% of the control group,while increase in the PMT transgenetic strains with 3.27 times higher than the control group.But the expression was inhibited in the interfering-expression strains,which differed from the situation of PMT gene.The expression of ODC increased in the over expression QPT and PMT strains,while decreased significantly in interfering expression strains.The expressions of CYP82E4v1 and CYP82E5v2 increased in PMT over expression strains,decreased in QPT over expression strains and inhibited in interfering expression strains.Compared with the control group,expression of CYP82E10 simultaneously decrease in the two treated groups,suggesting the inhibition effect of the QPT and PMT on CYP82E10.So as to say,the PMT can regulate the synthesis of nicotine and nornicotine.Then though constructing the interfering vector containing PMT and QPT,we can reduce the synthesis of nicotine effectively,so did the nornicotine.(3)The effects of CYP82E4v1 on the biosynthesis of nornicotineFor revealing the function of CYP82E4v1 gene involving in nornicotine biosynthesis in Nicotiana tabacum L.CYP82E4v1 genes of K326 were cloned by homologous cloning technology.Through the analysis of sequencing,CYP82E4v1 gene sequence is 1974 bp,including introns(420bp).CYP82E4v1 genetic similarity was 99.81% by blast in the NCBI,Only three bases are different,And encoding amino acid similarity was 100%.An Agrobacterium tumefaciens-mediated transformation method was used to transform tobacco cv.K326 with two plant expression vectors,pLF-35S-CYP,which contained CYP82E4v1-overexpressing fragment,and p LF-35S-CYPi containing interfered expression fragment,respectively.Sixty-one transgenic plants were acquired.The results of HPLC showed that the nornicotine content in CYP82E4v1 overexpressing plants was 88.9% higher than,that in interfered expression plants was 61.1% lower than that in the plants of cv.K326(WT).It indicated that the overexpression of CYP82E4v1 increased nornicotine content in tobacco,whereas the interfered expression of CYP82E4v1 gene inhibited nornicotine biosynthesis.The results of Real-Time PCR analysis indicated that the expression level of CYP82E4v1 gene in overexpressing plants was over 20 times the level in the CK,while that in interfered expression plants was only 6% of that in the CK.Meanwhile,the other nornicotine biosynthesis genes,such as CYP82E5v2 and CYP82E10,were suppressed to different extents in interfered expression plants.In addition,the “Gene-deletor” system contained recombinase gene FLP driven by the promoter of Arabidopsis thaliana senescent gene SAG12,which deleted the exogenous gene GUS?NPT II during the late stage of leaf development,was introduced into the mentioned expression fragments for the reason of acquisition of selectable-free transgenic tobacco plants with low nornicotine content.Meanwhile,study on directional mutation CYP82E4v1 gene by CRISPR/Cas9,We have 37 strains by hygromycin resistance,The next step will be the determination of nicotine content and mutation sequencing Analysisin in F1 plant,to obtain low nicotine in tobacco plants(4)Synthetic site on nicotine and nornicotineUsing UPLC-MS/MS technology and tissue culture technology,We analyzed the nicotine and nicotine synthesis position,including no root of shoot(shoot'),no stem leaf of root(root'),root and stem leaf of plant(root and shoot),callus.nicotine content results were obtained: the nicotine content in root was 34% lower than that in shoot,the nornicotine content in root was 34% lower than that in shoot,And root' was 9.75 times higher compared with shoot';the nornicotine content in root was 35.53% lower than that in shoot,And root' was 64.67 times higher compared with shoot'.The results of Real-Time PCR analysis indicated that the expression level of PMT in root was significantly higher than other,shoot' was only 0.14% of that in root'.Meanwhile,the other nornicotine biosynthesis genes,such as QPT,shoot' was 14.58% of that in root'.CYP82E4v1 gene expression was lowest all young tissue,the CYP82E5v2 gene expression that root' was 6.08 times lower compared with shoot',CYP82E10 gene expression that shoot' was 1.22 times lower compared with root'.So as to say,Nicotine and nornicotine can produce in All tobacco tissue as an important secondary metabolism substance,Most nicotine biosynthesis in root,but shoot is also testing the related gene expression,The results show that nicotine was accumulated in leaf through the root;And nornicotine was accumulated in leaf through the all tobacco part.(5)The effects of RNA interference of CYP82E4v1 on nornicotine content and Myzus persicae resistanceThe determination results of nicotine and nornicotine content via HPLC demonstrated that there was significant increase of nicotine content and reduction of nornicotine content in transgenic plants compared with those in wild-type plants.Exogenous application of IAA or GA3 could reduce the nicotine content in tobaccos,while ABA or 6-BA could increase the content of nicotine.And the more significant difference of nicotine content change in transgenic plants.Aphid-inoculation experiment demonstrated the number of aphid population in transgenic plants was significantly lower than wild-type plants at 12 d after aphid-inoculation.Meanwhile,the activity of AOEs and PAL in transgenic and wild-type tobacco plants after aphid-inoculation was measured.At 3 d after aphid-inoculation,both AOEs and PAL activity were significantly higher than controls,including wild-type plants with aphid-inoculation and transgenic plants with mock-inoculation.Also,the relative expression of these genes involved in salicylic acid/jasmonic acid(SA/JA)signaling pathways was analyzed at different stages after aphid-inoculation and the results demonstrated that there was significantly higher expression of JA-induced LOX gene in both transgenic and wild-type plants inoculated by aphid than the non-inoculated ones while no significant difference in the expression of SA-induced PR-1a gene among them was found,which indicated the JA-mediated resistance response was activated during aphid infestation.Moreover,although the expression level of BGL(another JA-induced gene)was less significant between the two inoculated tobaccos,it was significantly higher than the plant without inoculation,which was 1.4 and 2.2 folds higher than the non-inoculated controls respectively.To sum up,the improvement of aphid-resistance in transgenic tobaccos was based on nicotine accumulation which might cause nerve and antifeed toxicity and JA-mediated resistance response by enhancing the activities of AOEs and PAL.