Study On Cytological Basis And Molecular Regulation Of Rose Essential Oil Synthesis

Rosa rugosa Thunb.is an important ornamental and economical plant in the world.Rose essential oil extracted from its flowers is rich in aroma and has high health value.It is known as"liquid gold".The content of rose essential oil is very low,only about 0.3‰.It has important theoretical and practical value to explore the physiological and molecular mechanisms of rose essential oil synthesis and to improve the content and quality of rose essential oil through the genetic engineering.Flowers are the organs in which plants produce essential oil,and secretory epithelial cells or oil cells are the main sites to synthesize essential oil.The size of flower organs,especially the proliferation and expansion of epidermal cells,directly affect the production of essential oil.In this study,the developmental process of secretory epidermal cells of rose petals,the location of essential oil synthesis and accumulation,and the associated secretory pathway were studied by using Chinese R.rugosa 'Pingyin 1' as a test material.RrANT and RrAIL6 gene which related to cell proliferation of rose flowers were cloned and functionally identified with Petunia hybrida.The rose genetic transformation system was also established and optimized.Based on the preliminary research,RrNUDX1 which regulating the biosynthesis of main fragrance of rose flowers was transformed into R.rugosa to verify its function by infecting the somatic embryos with overexpression vectors.The aim of this study is to provide a theoretical basis for further indepth research on the physiological or molecular regulation of the main aromatic components in rose essential oil,and lay a foundation for creating new variety of R.rugosa with high essential oil content and promoting the development of rose industry.The main results are as follows:(1)The oil secretion mechanism of R.rugosa was initially identified.By using ultrathin sections combined with transmission electron microscopy,it was found that rose essential oil might be synthesized in two pathways:direct synthesis in plastids and conversion from starch grains.Then the endoplasmic reticulum initially processed a portion of osmiophilic oil for transport and reprocessing through the vesicles of the golgi apparatus.The energy for this process originated from the mitochondria,and the central large vacuoles were the space where the oils stored.Essential oils passed through the cytoplasmic membrane in two ways:through breaks in the cytoplasmic membrane and through exocytosis.(2)RrANT and RrAIL6 genes which related to flower size were cloned.The full-length cDNA sequence of RrANT and RrAIL6 was cloned by RACE method with R.rugosa 'Pingyin 1 'in large bud stage.The cDNA of RrANT is 2571 bp in length,with a complete open reading frame(1971 bp)which encoding 657 amino acids,the Genbank No.is MF802281.The cDNA of RrAIL6 is 1922 bp in length,with a complete open reading frame(1644 bp)which encoding a total of 548 amino acids,the Genbank No.is MK609564.(3)The expression patterns of RrANT and RrAIL6 in different developmental periods and different parts of the floral organs were studied.The results showed that the the change trend of expression level of RrANT and RrAIL6 were approximately the same in different developmental stages of organs by real-time PCR.The expression level was highest in the early developmental stage of organs and low in mature and senescence organs.This might be due to the fact that the high expression of RrANT and RrAIL6 can promote the cell proliferation in the early developmental organs.While in the mature organs,the process changed from cell proliferation to cell expansion,so the expression levels of RrANT and RrAIL6 were lower.However,the expression level of RrANT in the petals of the initial opening has increased rapidly.The expression patterns of RrANT and RrAIL6 were different in different parts of floral organs during full opening stage.The highest expression level of RrANT was in receptacles,followed by pedicles and calyxs,and the expressions in stamens,pistils and petals were very low.The highest expression level of RrAIL6 was in stamens,followed by receptacles,pistils and calyxs,and was lowest in petals and pedicles.In addition,subcellular localization showed that ANT protein of R.rugosa was targeted to the nuclear.(4)The overexpression vector of RrANT and RrAIL6 were constructed and their function were verified.The overexpression vectors of pC AMBIA1304-RrANT and pCAMBIA1304-RrAIL6 were successfully constructed and transferred into Petunia hybrida by Agrobacterium-mediated leaf disc transformation method.The transgenic plants were obtained and detected by GUS staining and PCR assay.Phenotypic observations of the RrANT transgenic plants revealed that overexpression of RrANT resulted in significant bigger flowers and leaves and increament of fresh weight of flowers compared to wild type.Further analysis showed that RrANT could promote cell proliferation to increase the number of cells.Transgenetic Petunia hybrida of RrAIL6 had not yet bloomed,and phenotypic observations need to be done later.(5)The genetic transformation system of R.rugosa was optimized based on the somatic embryo.The concentration of IBA in rooting medium was lmg/L.The OD600 value of Agrobacterium EHA105 to infect somatic embryos was 0.4 and the most suitable infecting time was 40min respectively.The concentration of Hyg resistance required for growth during 4 stages was also determined.At the same time,the RrNUDX1 gene,which related to the synthesis of main aroma components of rose essential oil was transferred to somatic embryos,and the RrNUDXl transgenic plants have been obtained.