Jasmonate Assisted Screening And Molecular Physiological Characterization Of Nicotine Synthetic Mutants Of Tobacco(Nicotiana Tabacum L.)

Tobacco is one of the most widely planted economic crops in China,it plays an important role in the national economy.Leaf quality is the decisive factor for economic benefits of tobacco.As an important chemical composition in tobacco leaf,nicotine,is closely related to tobacco quality and human healthy.Breeding of new cultivars with lower nicotine content is essential for tobacco breeding program.The traditional line selection and cross breeding had disadvantages as longer cycle,heavier workload and heredity of good characters being unstable,With the development of biological technology,it is necessary to screen nicotine mutants by using modern molecular biological techniques,reveal the molecular mechanism and combined with marker assisted breeding technology to speed up the breeding process of new tobacco varieties with lower inicotine contents.JA-signaling pathway displays an important role in regulating the genes of nicotine synthase enzymes,it is also a phytohormone that regulates seed germination and root elongation.These phenomena have potentials to be used for identification of regulators involved in the regulation of JA responses,nicotine synthesis.In this study,we screened nicotine mutants from an activation-tagged population of tobacco based on JA-sensitivity firstly,then analyzed molecular physiological characterization of these mutants,explore molecular mechanism of nicotine synthesis metabolic regulated by JA according to identify these mutation genes.The main results are as follows:1.Jasmonate assisted screening nicotine mutants of tobacco from activation-tagged population(1)We described a new method based on JA-sensitivity to screening nicotine mutants from an activation-tagged population of tobacco.In this approach,the mutants were first screened for abnormal JA responses in seed germination and root elongation,screened for nicotine content secondly;then rescreened out the nicotine mutants maintaned stable JA sensitivity and nicotine contents within 3th generation.The method is simple,effective and low-cost,and the finding of transcriptional changes of nicotine synthetic genes in the mutants shows potentials for identifying novel regulators involved in JA-regulated nicotine biosynthesis and pathogenresistance.(2)We identified 48 lines with abnormal JA responses in both germination and root elongation from 3000 T1 activation-tagged population of tobacco,then obtained 5mutants which maintained stable nicotine contents and JA responses for three generations in further screening.These mutants were named nit3,nit6,nit7,nit9 and nit10,among them: nit3 and nit6 showed low JA sensitivity and decreased nicotine contents,nit7 showed JA sensitivity and increased nicotine content,nit9 and nit10 showed high JA sensitivity and high nicotine contents.We also successfully obtained 3special nicotine mutants that maintained stable nicotine contents and JA responses for three generations in further screening.These 3 mutants were named nit11,nit14 and nit17,nit11 showed decreased nicotine content before head cut and increased nicotine content after head cut,it showed JA insensitivity also,nit14 showed decreased nicotine content before head cut and high nicotine content after head cut,it showed low JA sensitivity,nit17 showed high nicotine content before head cut and decreased nicotine content after head cut,it showed high JA sensitivity.2.Physiological characteristics of mutantsTaking the 5 nicotine mutants and 3 special nicotine mutants as materials,we measured the enzymes of carbon and nitrogen metabolism(INV,GS)before and 1,14,28 days after treatment with MeJA and head cut,then measured the soluble sugar content,total nitrogen(N)content hormone(ABA,IAA,GA,ZR)contents and photosynthetic indexes(chlorophyll content,net photosynthetic rate,intercellular CO2 concentration,stomatal conductance)before and 28 days treatment with MeJA and head cut in the mutants.The main results are as follows:(1)The nicotine contents of the 5 nicotine mutants had a negative correlation with INV and GS activities,while had a positive coorelation with total nitrogen content.After MeJA treatment and head cut,GS activity decreased significantly and the higher nicotine content,the higher total nitrogen content.The nicotine contents had a positive correlation with ABA content,while had a negative coorelation with IAA and GA contents.After MeJA treatment and head cut,IAA and GA contents decreased significantly.The nicotine contents has a negative correlation with chlorophyll content,net photosynthetic rate and intercellular CO2 concentration,while had a positive coorelation with stomatal conductance.Stomatal conductance of the 5 nicotine mutants increased significantly after MeJA treatment and head cut.(2)The 3 special nicotine mutants had abnormal nicotine content changes before and after head cut,and they showed different physiological characteristics from the 5nicotine mutants.The nicotine content of nit11 increased significantly after head cut,which was probably caused by increased soluble sugar content or increased ABAcontent or decreased chlorophyll content and net photosynthetic rate.The nicotine content of nit14 increased significantly after head cut,which was probably caused by increased GA content or stomatal conductance.The nicotine content of nit14 dncreased after head cut,which was probably caused by increased total nitrogen content or decreased soluble sugar content or decreased IAA and GA content or decreased chlorophyll content and net photosynthetic rate or increased stomatal conductance.3.Molecular characteristics of mutants(1)Identification of the 8 mutants by Bar gene PCR revealed that the activation-tagging pSKI015 had integrated in the mutants genome.The result of southern blot showed that the nicotine mutants nit6 and nit9 were inserted 2 copies of T-DNA,nit3,nit7 and nit10 was inserted 1 T-DNA.Special nicotine mutant nit11 were inserted 2 copies of T-DNA,nit14 and nit17 was inserted 1 T-DNA.(2)JA-sensitivity of the 8 mutants were quite consistent with the expression levels of NtMYC2 gene.(3)The expression level of nicotine synthesis genes in the 5 nicotine mutants were regulated by JA,it wasis consistent with the mechanism of nicotine synthesis induce by JA.The expression level of nicotine synthesis genes in the 3 special nicotine mutants were regulated by JA also,but each of these mutants had different expression model.nit11 showed lower expression level of NtBBL in the absence of MeJA,and xhibited higher expression levels of NtBBL and lower expression levels of NtPMT after MeJA treatment.nit14 showed lower expression level of NtODC and higher expression level of NtA622 before and after MeJA treatment,and showed higher expression level of NtPMT before MeJA treatment,after MeJA treatment,the expression levels of NtPMT,NtBBL decreased and the expression levels of NtNUP1 increased.Almost all of the expression levels of nicotine biosynthesis genes of nit17 were not different from the control before and after MeJA treatment,but the expression lvel of its nicotinic transporters gene Nt NUP1 was obviously lower than that in control before and after MeJA treatment.4.Flanking sequences amplification and candidate gene analysis of mutants(1)Using FPNI-PCR,TAIL-PCR,single primer PCR,IPCR and plasmid rescue to amplify flanking sequences of the 8 mutants.The optimized FPNI-PCR had the best amplification results and it could be the method to amplify flanking unknown DNA sequences of tobacco.(2)Obtained 10 different flanking sequences totally,the pathogenmutants nit11,nit14 and nit17 could find homologous proteins after sequence blast in tobacco genetics database.nit11 had 2 genes near T-DNA insertion site,they were named Ntab0396680 and Ntab0396670 in tobacco genetics database.nit14 had 2 genes near T-DNA insertion site,they were named Ntab0583900 and Ntab0583910 in tobacco genetics database.nit17 had 4 genes near T-DNA insertion site,they were named Ntab0555610,Ntab0555610,Ntab0555610 and Ntab0555610 respectively in tobacco genetics database.(3)An unique candidate gene was identified correspondingly from nit11,nit14 and nit17,the candidate gene number of nit11 was D1-2,named as Ntab0396670;the candidate gene number of nit14 was D4-1,named as Ntab0583900;the candidate gene number of nit17 was D7-1,named as Ntab0555610.(4)The result of JA response analysis showed that the candidate gene expression level of nit17(Ntab0555610,D7-1)increase lightly after MeJA treatment.5.Identify the candidate gene of nit17(1)Taking the co-segregation analysis to confirm whether nit17 were caused by T-DNA insertion.The result showed that the mutation phenotype of nit17 was caused by T-DNA insertion,the mutation phenotype separated in the offspring.The mutants which showed mutation phenotype exist heterozygous genotype in the same lines,these results indicate that nit17 was controlled by dominant single gene.(2)The candidate gene(Ntab0555610)of nit17 was similar to pleiotropic drug resistance protein 1(PDR1),we named it as NtPDR30.Constructed tranGSenic tobacco lines that over-expressed NtPDR30 gene with eGFP,the tranGSenic lines and nit17 could reproduced mutation phenotype after inoculated EDC.(3)The results of homology comparison in NtPDR30 and other PDR protein from different species showed that these proteins had high homology.NtPDR30 form a specialized structure in ABC transporters and called“NBD-TMD-NBD-TMD”.The result of subcellular location showed that NtPDR30 expression on the plant cell membrane.(4)By analysising the gene expression level of NtPDR30 in different tissues before and after MeJA treatment from common tobacco“honghuadajinyuan”(HD),the result showed that NtPDR30 could induced by MeJA in tobacco roots.(5)The nicotine contents of nit17 and T0 decreased after MeJA treatment and inoculated EDC.But the nicotine synthesis genes of nit17 changed almost nothing,we hypothesized that the nicotine contents decreased of nit17 caused by nicotinetransport.NtPDR30 may suppressed nicotine transportation.(6)There was a close and intrinsic relationship between nicotine metabolism and pathogens resistant regulated by MeJA.