Analysis On The Formation Mechanism Of Fresh-Sweet Aroma In Tobacco Mutant 8

Plant aroma is a comprehensive description about the plant volatile gases by human olfactory senses.Each plant has a unique aroma,which is closely related to the type and composition ratio of volatile organic compounds(VOCs).The research on the formation mechanism of plant aroma is very slow for the diversity of volatile compounds and the difficulty in detecting trace compounds.In earlier,we obtained a tobacco mutant T8,which upper young leaves had obvious fresh-sweet aroma at bud stage.The trait was identified as a single gene recessive mutation by population genetic analysis,but the related genes have not been identified,and the formation mechanism of fresh-sweet aroma have been uncover.Here,we focused on T8 and CK(ZY100),and tried to uncover the formation mechanism of fresh-sweet aroma based on non-targeted volatile metabolites detection,transcriptome sequencing,gene family analysis,gene co-expression network construction,gene identification and so on.The main results are as follows:1.In the bud stage of T8,the volatile gases from its upper leaves were collected in situ in the field.The GC-MS results showed that the volatile gases contained 26 compounds,among which 18 were terpenes,the relative content of which was 71.74%,2.34 times that of the control.The relative content of linalool was 52.12%,3.4 times that of the control,which was the highest relative content and the most significant difference.Linalool synthase(LINS)gene belongs to the TPS gene family.Transcriptome sequencing was performed in the upper and middle leaves of T8 and ZY100 at three developmental stages.Results showed that differential genes were mainly focused at pathawys like plant hormone signal transduction pathway,secondary metabolite synthesis pathway,and terpene synthesis pathway,and so on.2.Fifty-five TPS genes were identified from the TN90 genome,which could be subdivided into five subfamilies(a,b,c,e/f and g),with the number of members being 20,18,8,8 and 1,respectively.The length of TPS ranged from 191 to 865 aa,and the isoelectric p I ranged from 4.88 to 8.46.TPS mainly located in the cytoplasm,plasma membrane and nucleus.Thirty TPSs contained both conserved motifs “RR(x)8W” and “DDxx D”.The known LINS genes were usually members of TPS-b and TPS-g,and LINS candidate gene set will be gradually constructed.3.Based on public transcriptome,I analyzed the expression pattern of TPS gene in various tobacco tissues.The results showed that the number of TPS genes specifically expressed in stem,leaf,flower and glandular hair was 3,3,3 and 2,respectively.Four TPS genes were expressed in all tissues.Five TPS genes were involved in the aging process of leaves and flowers.In glandular hair,flowers and leaves,there were 10 members of TPS-b subfamily with high expression,such as Nt TPSb01～Nt TPSb04,Nt TPSb10～Nt TPS15 and Nt TPSb16.And the ten TPS genes were added into the LINS candidate gene set.Based on public transcriptome,I analyzed the expression pattern of TPS genes respond to various environmental stress.The results showed that 17 TPS genes did not respond to any environment stress.The number of TPS genes in response to cold,drought,topping,bacterial blight infection,CMV infection,salt stress and cadmium ion stress were 24,12,13,24,8,0,5,respectively.There are 6members of TPS-b subfamily responding to topping,such as Nt TPSb01,Nt TPSb06,Nt TPSb08,Nt TPSb10,Nt TPSb11 and Nt TPSb17.And Nt TPSb06,Nt TPSb08 and Nt TPSb17 were added into the LINS candidate gene set.4.The paper obtained 141 terpene pathway genes,1445 TFs,238 HRGs,238 ABCs and 107 PSs from TN90 genome annotations,and constructed a co-expression network of MVA/MEP pathway based on T8’s transcriptomes.The results showed that the key enzyme genes upstream of MVA/MEP pathway were mainly positively correlated with TPS genes.The connection degrees between MVA/MEP pathway and TFs/ HRGs/ ABCs/ PSs was 4283,713,787 and 881,respectively.It was mainly positively correlated with the first three regulation factors,while mainly negatively correlated with PSs.There are7 members of TPS-b or TPS-g subfamily,such as Nt TPSb01,Nt TPSb10～Nt TPSb12,Nt TPSb15,Nt TPSb16,Nt TPSb18 and Nt TPSg01.And Nt TPSb15,Nt TPSb18 and Nt TPSg01 were added into the LINS candidate gene set.5.Base on LINS candidate gene set and expert analysis,11 candidate LINS genes(Nt LINS01～Nt LINS11)were obtained.The q PCR results showed that Nt LINS01 was up-regulated in young leaves,flower buds,young flower,mature flower and glandular hairs;Nt LINS05 was up-regulated in young leaves,flower buds and glandular hairs.The paper cloned Nt LINS05 gene,which contains 7 exons.The gene length and cds length of Nt LINS05 were 5784 bp and 1842 bp,respectivly.The protein encoded by Nt LINS05 contains 613 aa with a molecular weight of 70.14 k Da and a theoretical isopotential point of6.20,and is located in the cytoplasm.The in vitro result showed that Nt LINS05 was efficient to synthesize linalool using GPP as substrate.6.Hypothesis of the formation mechanism of T8’s fresh-sweet aroma.TFs,HRGs and ABCs of T8’s fresh leaves are are activated to reguate terpene pathway under the induction of suitable sunlight.then some genes of terpene pathway are upregulated in a synergic way,and the total amount of VOCs is comprehensively increased and released through stomata,forming fresh-sweet aroma,in which linalool is main component.When sunlight is excessive,PSII activates the mechanism of nonphotochemical quenching(NPQ),upregulate the expression of PSs,and emits excessive light energy in the form of thermal energy.At the same time,tobacco leaves would close stomatas to reduce transpiration,and VOCs could not pass through stomatas,and the fresh-sweet aroma would weaken or disappeared.