Expression Analysis Of Transcription Factor RrMYBS4 And Its Targeted Regulatory Gene Rr11690 In Response To Hormones In Rose Rugosa

As common horticultural crops,Rose rugosa and Rose chinensis have been widely loved because of their diverse colors and exotic flowers fragrance.Because of their ornamental value and economic value.Studies have shown that the composition of fragrance in Rose rugosa and Rose chinensis is complex and diverse,mainly including terpenoids,benzene/phenylpropanoids and fatty acid derivatives.The change of composition and proportion of these substances is an important factor affecting the formation of fragrance.ATP binding cassette（ABC）transporters are widely involved in the transport of plant secondary metabolites,such as some terpenoids and phenylpropanes.As a large family,MYB transcription factors participate in hormone response and play an important role in rose fragrance biosynthesis pathway.In the early stage,the research team found that 1R-MYB transcription factor RrMYBS4 in rose is located in the nucleolus,can regulate ABC transporter coding gene Rr11690,and bind to the promoter of the latter,which may be involved in regulating the transport of volatiles.Therefore,this study further explored the function of RrMYBS4.Through applying plant hormones,drought or darkness treatment on wild type Rose rugosa or Rose chinensis resources and transgenic Arabidopsis thaliana,and combining RT q PCR technology and GUS histochemical staining to detect the expression of RrMYBS4 and Rr11690,we obtained environmental factors that may affect the expression of RrMYBS4,and the role of rose in the synthesis and release of floral aroma substances was studied through its original transformation.It lays a foundation for comprehensively analyzing the biosynthetic pathway of rose fragrance.The research results are as follows:1.The overexpression vector and interference vector of RrMYBS4 were constructed,and the genetic transformation of Rosa rugosa‘Bao White’was carried out.The expression of RrMYBS4 was detected by RT q PCR,and five RrMYBS4-RNAi interference strains were obtained.All plants are currently in the vegetative growth stage,and there is no significant difference in plant growth status and leaf phenotype compared with the control group.2.The acting elements of the promoters of RrMYBS4 and Rr11690 were analyzed for hormone and stress treatment.According to the analysis results,the wild type Rose rugosa and Rose chinensis materials were treated with ABA,GA,Me JA and other hormones,as well as drought and dark treatment.The results showed that the expression of RrMYBS4 in Rosa rugosa‘Zizhi’treated with Me JA decreased significantly within6-18 h,and returned to the same expression level as the untreated plants after 48 h;Rr11690 significantly increased within 12-48 h after Me JA treatment;The expression level of Rr11690 increased 6 h after rapid propagation of Rosa rugosa‘Zizhi’seedlings treated with GA.The expression level of Rc MYBS4 and Rr11690 homologous genes in Rose chinensis increased 6 h after ABA and GA treatment.3.The expression vector of fusion GUS reporter gene of RrMYBS4 and Rr11690promoter fragment was constructed,and Arabidopsis thaliana and Rosa rugosa‘Bao White’were transformed respectively.Four high expression lines were identified by GUS staining of the T₁generation Arabidopsis inflorescence.The collected T₂generation seeds were stained for temporal and spatial expression for 5 days,10 days and 15 days.The staining area of the transgenic RrMYBS4pro::GUS positive seedlings was basically limited to the epidermal hairs of the real leaves.The transgenic Rr11690pro::GUS positive seedlings had obvious staining in the root vascular,and some staining was also found on the leaves and hypocotyls.According to the elements on the promoter,the transgenic Arabidopsis thaliana was treated with hormones and stress.The root stain of Rr11690pro::GUS positive seedlings treated with 0.1 mmol/L ABA was significantly darker than that of 6 h at 12 h,but the root tip was not stained.The dyeing effect of other treatments had no obvious change.In conclusion,through quantitative analysis of the effects of hormone and environmental stress treatment,appearance analysis of transgenic roses,and heterologous verification in Arabidopsis,the study preliminarily explored the possible stress conditions that MYB transcription factor RrMYBS4,which is related to the transport of rose fragrance compounds,and Rr11690,which is targeted for regulation,might respond to,providing ideas for the creation of new rose fragrance varieties.