Preliminary Study On Creating High-fragrance Petunia By Molecular Breeding

Floral scent is a very important horticultural character of flowers.Flowers VOCs play an important role in attracting insects,promoting pollination,and coping with exogenous damage^[1].The attraction of pollinators is mainly mediated by benzene-containing compounds,while the defense function is mainly mediated by terpenes and benzene VOCs^[3].For humans,floral fragrance is also an important horticultural trait with strong aesthetic and emotional value^[2].Volatile organic compounds from flowers are widely used in the production and manufacturing of perfume,daily chemical products,condiments and medicines^[1].The floral scent compounds mainly include terpenoids,benzene compounds and fatty acid derivatives.Their biosynthesis pathway is a very complex network with many overlapping,overlapping and competitive relationships.On the basis of the research on the key genes of floral synthesis pathway,directional breeding can be carried out by using molecular breeding methods,which can achieve more accurate regulation than traditional breeding methods.In order to obtain a new scent of petunias,our study based on the floral synthetic approach,the research of the jasmine flowers scent is built on synthesis of key genes driven by specific promoter,and conversion of petunias,screening of positive transgenic plants,the flora scent compounds,phenotypic records and determined,and further tested the scent biosynthesis pathway in genetically modified materials of structural genes and transcription factor expression level changes.The main results are as follows:1.Flower-specific expression vector construction.Use a flower-specific promoter CbLIS from Clarkia breweri.repalced the 35S promoter on vector pK7FWG2.0 by double enzymes restriction and adapter ligation.The key gene of jasmine floral scent synthesis JsTPS4 was constructed by homologous recombination,get a pK7FWG2.0-proLIS:JsTPS4 vector.2.PK7FWG2.0-proLIS:JsTPS4 vector was transformed into Agrobacterium GV3101,then transformed to petunia by leaf disk method.Total 33 transgenic materials were obtained by kanamycin selecting.There was no significant difference in plant growth between transgenic materials and wild types.The leaves of some transgenic materials were wrinkled,and the flowers of some transgenic material were wrinkled or proliferated.A total of 28 positive transgenic plants were identified by PCR in T0 generation.Selecting by kanamycin,total 53 T1 generation were obtained.We obtained 2 positive transgenic plants in Line 22-7,21 positive transgenic plants in Line44-1 and 13 positive transgenic plants in Line 66-1 by PCR test.The expression of JsTPS4 was significantly increased.The expression of JsTPS4 at 2 d 23:00 was 20 to 800 times higher than wild typ e.The expression of JsTPS4 at 3 d 23:00 was 25 to 500 times high er than wild type.3.Identification and content analysis of floral scent components in transgenic materials.The floral scent compounds in the flowers of transgenic materials were extracted with n-hexane?UPLC Grade?.The results of GC-MS showed that the contents of methyl benzoate and benzyl benzoate were significantly different from wild type.The content of methyl benzoate was 1.21 to 3.94 times to wild type,and the content of benzyl benzoate was 1.57 to 14.35 times to wild type.The total floral scent weight was 1.09 to 1.38 times to wild type.The content of benzyl alcohol increased in some transgenic strains,and its contents was 3.03 to 8.39 times to wild type.4.Based on the phenomenon of significant increase in benzene content in floral scent components of transgenic materials,in this study,the expression patterns of eight structural genes?PhEPSPS,PhCM1,PhADT,PhPAL,PhPAAS,PhKAT1,PhBPBT,PhBMST1?and three transcription factors?PhODO?,PhEOB?,PhEOB??related to benzene synthesis were investigated.The expression of upstream genes?PhEPSPS,PhCM1,PhADT?of phenylalanine synthesis in benzene synthesis pathway was higher than wild type.The expression of downstream genes?PhPAL,PhPAAS,PhKAT1,PhBPBT,PhBMST1?varies in different transgenic strains.Based on the obvious changes in floral scent compounds of Line 22-7 and Line 44-1,the expressions of PhPAL,PhPAAS,PhKAT1,PhBPBT and PhBMST1 also showed an upregulated trend compared with wild type.The expression of transcription factor PhODO?show a certain correlation with PhEPSPS and PhPAL.The expression of transcription factors PhEOB?and PhEOBII was not consistent with the expression of PhPAL.Therefore,PhODO?may be the key gene affecting the expression of PhEPSPS.In this study,we obtained several transgenic petunia materials with significantly improved scent compouds methyl benzoate?1.21 to 3.94 times?and benzyl benzoate?1.57 to 14.35 times?were obtained,which provided breeding materials for improving flower scent in further study.