Study On The Expression Characteristics And Molecular Regulation Of Genes Related To Labdane Diterpenoids Biosynthesis

This study analyzed the differences of composition and content of labdane-diterpenoid in glandular trichomes secretions of different types of tobacco varieties and their ancestors,and used real-time quantitative PCR technology to study the expression patterns of genes related to labdane-diterpenoid synthesis among different types of tobacco cultivars,as well as among different tobacco tissues.The expression and regulation mechanisms of labdane-diterpenoid synthase genes were further explored through the use of overexpression techniques and gene knockout methods.The main findings are as follows:1.The composition and content of major diterpene secretions in different types of tobacco cultivars and two ancestors were analyzed by GC-MS.The results show that the cembrane-diterpenoid has higher contents in various tobacco varieties;while the labdane-diterpenoid only existed in the Basma YNOTBS1(Oriental tobacco)and Beinhart 1000-1(Cigar)and Yuyanll(Flue-cured tobacco);among the two ancestors,N.sylvestris contains only cembrane-diterpenoid,while the N.tomentosiformis contains only labdane-diterpenoid.2.The 3D microscope with super wide depth of field was used to observe the trichomes’ morphology of different types of tobacco varieties.The results showe that there are differences trichome-types between two ancestry species,N.sylvestris tobacco contains three types of trichomes,ie,tall glandular trichome,short glandular trichome and protective trichome;while N.tomentosiformis contains two trichome-types,namely tall glandular trichome and short glandular trichome.The types of glandular hairs contained in five different types of N.tabacum varieties are not exactly the same.Among them,flue-cured tobacco K326 and Yuyan 11 were similar to N.sylvestris and contained three types of glandular hairs;The cigars Beinhart 1000-1 and Burley Tobacco Dabai 1 are similar to N.tomentosiformis tobacco and contain two types of glandular hairs,namely tall glandular trichome and short glandular trichome.3.Analysis of the expression level of labdane-diterpenoid synthase gene in flue-cured tobacco K326 tissues.The results showe that labdane-diterpenoid synthesizing genes NtCPS2 and NtABS have higher expression levels in glandular trichomes,while have no expression or very low expression in other tissues,this indicates that these two genes are specifically expressed in glandular trichomes.The expression of NtCPS2 gene are different among different tobacco varieties.The specific expression was that NtCPS2 gene have the highest expression level in Basma YNOTBS1,and Beinhart 1000-1 and Yuyan 11 were the second,K326 and Dabai l are the lowest.4.The NtCPS2 promoter and NtABS promoter sequences were obtained from the NCBI gene database.Bioinformatics analysis reveale that the NtCPS2 promoter sequence contains an optical signal response element,an abscisic acid response element,a gibberellin response element,and circadian rhythm control.At the factor binding site,the NtABS promoter sequence contains cis-acting elements that respond to light,high temperature,drought,gibberellin,etc.,and regulatory elements that are involved in physiological regulation control,this is suggesting that these role elements may be involved in the expression regulation of NtCPS2 and NtABS genes.The NtCPS2 gene sequences in Basma YNOTBS1,K326,and 8306 were compared with the reported sequences in the NCBI database.The results showe that the sequences of the three genes are identical to those in the database.Phylogenetic tree analysis showe that the relationship between NtCPS2 and N.benthamiana in N.tabacum are greater than other solanaceous species such as potato and tomato.5.In order to further improve the tobacco leaf quality of flue-cured tobacco K326,the recombinant plasmid of the NtCPS2 gene and pCAMBIAI1305 overexpression vector fragment was constructed by the enzyme-cut method.Finally,the target gene was introduced into flue-cured tobacco K326 by agrobacterium tumefaciens-mediated method.A total of 10 regenerated plants were obtained through screening and differentiation.The next step was to identify the positive plants,and to determine the content of diterpenoids in the flue-cured tobacco K326 and observe the phenotype of the plants.6.In order to further improve the specialty flue-cured tobacco Yuyan 11,this study selected an exon region sequence of the NtCPS2 gene and designed a gRNA sequence according to the working principle of the CRISPR/Cas9 gene editing system.The gRNA sequence and 35S:hspCas9:U6:gRNA vector were constructed into a recombinant plasmid,and the NtCPS2 gene of flue-cured tobacco 8306 was knocked out by agrobacterium tumefaciens-mediated method.T1 generation sequencing of transgenic positive plants revealed that a single base insertion occurred at base 324 of the NtCPS2 open reading frame,leading to early termination of the encoded peptide chain.The homozygous mutant plants were observed for plant growth,glandular hair morphology,density statistics,and foliar chemical composition.The results showed that mutations in the NtCPS2 gene did not affect plant growth and glandular trichome density,but affected glandular trichome head size.Foliar chemical composition test results showe that the glandular hair secretions of mutant homozygotes lacked the labdane-diterpenoid component.