| Paeonia section Moutan DC.are famous traditional and precious flower in China,has high ornamental value with large and beautiful flowers.‘High Noon’,as a late flowering and yellow flower cultivar of tree peony,has important application value because of its late flowering,pure color and strong floral fragrance.Plant volatiles are a variety of volatile components synthesized by plants and released into the air.Flower volatiles,mainly derived from a variety of volatile components produced in flower,is one of the important quality indicators of tree peony.In previous studies,some progress has been made in the research on the floral aroma of ornamental plants,but the research and application in tree peony is still lacking.In this study,the regular patterns of flower volatile composition and relative content were analyzed and some terpene synthase genes were cloned and identified in tree peony cultivar ‘High Noon’.The main results are as follows:1.A total of 34 volatile substances compounds were detected in ‘High Noon’ and the sequences of the total relative content of the volatiles in different flowering periods was as follows: full blooming > initial flowering > decay period> blooming stage,and the highest relative content was in petals.The changes during one day showed an increasing firstly and then a decrease,and reached the peak between 14:00-16:00.The linalool,2,6-Octadiene-1-diol,3,7-dimethyl,germacrene D were the main floral ingredients in the volatile of tree peony cultivar ‘High Noon’.2.The Linalool synthase(Ps LIS)gene and Germacrene D synthase(Ps GDS)gene were cloned and identified from tree peony cultivar ‘High Noon’.The open reading frame of Ps LIS is 1698 bp,encoding 565 amino acids,and Ps GDS is 1725 bp,encoding 574 amino acids.Ps LIS and Ps GDS are labile hydrophilic,nontransmembrane and non-secretory proteins and with a conserved terpene cyclase domain.It was predicted to be localized in the cytoplasm.The phylogenetic analysis revealed that Ps LIS and Ps GDS has a close evolutionary relationship with woody plants,but distantly related to herbs.3.The expression level of Ps LIS and Ps GDS in petals were significantly higher than calyx,stamen and pistil.Ps LIS was expressed highest at the petals of different flowering periods.and Ps GDS was expressed highest at the initial flowering stage.The expression of Ps LIS was highest at the time period from 14:00 to 16:00 and Ps GDS was from 12:00 to 14:00 during one day.The expression pattern of Ps LIS and Ps GDS was consistent with the relative release of linalool and geraniene D respectively,and showed significant positive correlation.It is speculated that Ps LIS and Ps GDS may be the key genes controlling the volatilization of linalool and geraniene D in tree peony cultivar ‘High Noon’.4.Subcellular localization experiments showed that Ps LIS and Ps GDS were mainly localized in the cytoplasm,revealing its plays catalytic function in the cytoplasm.5.The 35S::Ps LIS and 35S::Ps GDS expression vectors were constructed and transformed into Agrobacterium,and were carried out to get genetic transformation tobacco by leaf disk method.Three Ps LIS and three Ps GDS genetic transformation tobacco positive lines were obtained respectively.The relative expression of Ps LIS and Ps GDS in genetic transformation tobacco were higher than wild type,and the highest expression level of Ps LIS and Ps GDS were 4.92 and 5.2 times to wild type respectively.In summary,this study revealed the emitted pattern analysis of flower volatiles in tree peony cultivar ‘High Noon’.The gene Ps LIS and Ps GDS were cloned and carried out bioinformatics analysis,expression pattern analysis and subcellular localization analysis.The genetic transformation tobacco with overexpression of Ps LIS and Ps GDS gene were obtained.These results lay the foundation for further analyzing the molecular mechanism of flower volatile substances production and different fragrance types breeding of tree peony. |
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