Molecular Identification And Functional Study Of Jasmine (Jasminum Sambac(L.) Ait) Benzyl Acetate Synthase

Jasmine [Jasminum sambac(L.)Ait] is a plant of the genus Oleaceae Frangipani.Jasmine is a world-famous spice crop due to its unique and rich floral fragrance.In China,jasmine tea made from double petaled jasmine as the raw material is very popular among consumers,and the low utilization rate of jasmine aroma is the main reason for the high cost of jasmine tea.The current research on the aroma of jasmine mainly focuses on the extraction of essential oils,In terms of aroma component determination and production process improvement,there is little research on the molecular mechanism of jasmine fragrance release and metabolic regulation.Previous studies have found that the three highest content of jasmine aroma components are linalool,α-farnesene and benzyl acetate,and benzyl acetate is the main component of aroma absorption when scenting jasmine tea.Benzyl acetate has a characteristic "sweet fruity fragrance",which is an important fragrance ingredient.The content of benzyl acetate is one of the important quality indicators of jasmine essential oil in India.Benzyl acetate is formed by the benzyl alcohol acetyltransferase encoded by the BEAT subfamily of the BAHD gene family,which catalyzes the transfer of the acetyl group of acetyl-Co A to benzyl alcohol.This thesis conducts basic research on the molecular mechanism and metabolic regulation of the synthesis of benzyl acetate,hoping to lay the foundation for the molecular breeding of high-scent jasmine.The main contents include: screening of Js BEAT-related genes,functional verification of Js BEAT-related genes,and searching of Js BEAT upstream regulatory factor genes.The main findings are as follows:1.Two Js BEAT candidate genes were screened and their expression dynamics were highly correlated with the content of benzyl acetate in the petals.The content of benzyl acetate in the petals of Jasminum sambac was determined during the opening process.It was found that the content of benzyl acetate was higher in 3h-9h after jasmine bloomed,and then gradually decreased.Rhythm.Nine Js BEAT genes were screened from the Jasmine transcriptome database.The petals at different time points of jasmine blooming were used as materials.Through fluorescence quantitative RT-PCR analysis,it was found that the relative expression of Js BEAT1 and Js BEAT2 genes was the highest at 6h-9h after jasmine blooming.,Consistent with the release law of benzyl acetate,identified Js BEAT1 and Js BEAT2 genes as the research objects.Through RT-PCR detection of genes in different tissues,it was found that the relative expression level of Js BEAT gene in petals was the highest,and the relative expression level in stems and leaves was lower.2.A series of Js BEAT1 and Js BEAT2 expression vectors were constructed,and the subcellular localization of the encoded protein in petal protoplasts was clarified.Blast the full-length CDS of Js BEAT1 and Js BEAT2 from the Jasmine transcriptome database,and cloned the gene sequences of Js BEAT1 and Js BEAT2 to be 1347 bp and 1338 bp,respectively.The Agrobacterium-mediated tobacco transient transformation system and PEG-mediated jasmine petal protoplast transformation System,found that the subcellular localization of jasmine Js BEAT1 and Js BEAT2 genes are mainly in the cytoplasm and plasma membrane.3.Revealed the enzymatic characteristics of Js BEAT1 and Js BEAT2.A prokaryotic expression vector was constructed,and recombinant Js BEAT1 protein and recombinant Js BEAT2 protein were induced and purified in vitro,both of which can catalyze the substrate benzyl alcohol and acetyl-Co A to produce benzyl acetate.The Km value of the recombinant Js BEAT1 protein to the substrate benzyl alcohol is 447.3μM,the Km value of the recombinant Js BEAT2 protein to the substrate benzyl alcohol is 278.7 μM,and the Km of the Js BEAT1 to the substrate benzyl alcohol is 1.6 times that of Js BEAT2,indicating that Js BEAT2 has a stronger ability to bind to the substrate benzyl alcohol.The Km value of the recombinant Js BEAT1 protein to the substrate acetyl-Co A is546μM,the Km value of the recombinant Js BEAT2 protein to the substrate acetyl-Co A is 317.3μM,and the Km of the Js BEAT1 to the substrate acetyl-Co A is 1.7 times that of Js BEAT2,indicating that the Js BEAT2 recombinant protein The ability to bind to the substrate acetyl-Co A is stronger.4.Verify the function of Js BEAT1 and Js BEAT2 genes through a heterologous stable expression system.In order to verify the function of jasmine Js BEAT1 and Js BEAT2 genes in the synthesis and metabolism of benzyl acetate in plants,since jasmine has not established a stable genetic transformation system,the heterologous genetic transformation system of the model plant petunia is selected.Petunia has the ability of benzyl acetate.Anabolic pathway.The overexpression vector of flower-specific promoter LIS was constructed,and petunia was transformed by Agrobacterium-mediated leaf disc method.Finally,3 positive lines of T2 generation of Js BEAT1 and 4positive lines of T2 generation of Js BEAT2 were screened.Fluorescence quantitative RT-PCR detection of transgenic petunia and wild type showed that the relative gene expression of Js BEAT1 and Js BEAT2 transgenic lines increased by 23.53-35.44 times and 23.53--50.97 times,respectively,compared with wild-type.The content of benzyl acetate in the petals of transgenic petunia and wild-type petunia was determined,and it was found that the benzyl acetate content of Js BEAT1 and Js BEAT2 transgenic lines were increased compared with that of wild-type petunia,by 2.06 times～3.27 times,2.33 times～ 3.83 times.In phenotype,it was observed that some transgenic plants showed thickening of leaf epidermis,abnormal shape and convexity,and shrinkage after the flowers opened.This may be due to the interference of Agrobacterium-mediated plant growth.The transgenic plants are apparently similar to wild-type plants.5.Explained the potential regulatory mechanism of Js BEAT1 and Js BEAT2 and candidate genes through the bio-infographic analysis and experimental analysis of the promoter.The promoter sequences of Js BEAT1 and Js BEAT2 were cloned by chromosome walking method.The lengths were 1474 bp and 1570 bp,respectively.Analysis of functional elements revealed that the promoters of Js BEAT1 and Js BEAT2 genes contained light response,hormone response,anaerobic response,rhythm,and MYB binding Components and other related regulatory elements.ACE,Sp1,TATC-box,TCCC-motif,chs-CMA2 a,CAT-box,CCAAT-box are unique to the promoter region of Js BEAT1,while ARE,GT1-motif,I-box,LTR,MBS,and p-box are The uniqueness of the promoter region of Js BEAT2 indicates that different gene members of Js BEAT may have functional differences in resistance regulation and hormone response.The promoter of Js BEAT1 gene contains elements related to the binding of MYB,MRE,CCAAT-box and MYB,and the promoter of Js BEAT2 gene contains elements related to the binding of MBS,MYB,MRE and MYB.It is speculated that the promoter of Js BEAT gene can be associated with MYB-related transcription factors.Combines with regulating floral fragrance.The Js MYB transcription factor was screened from the transcriptome database,and the Agrobacterium-mediated tobacco co-transformation transient expression system was used to verify the activation of Js MYB305,Js MYB86,Js MYB108,and Js LHY on the promoters of Js BEAT1 and Js BEAT2.It was found that the effect of Js MYB in the tobacco system,Js MYB108 has the strongest activation effect on the Js BEAT1 promoter,followed by Js LHY;Js LHY has the strongest activation effect on the Js BEAT2 promoter.The inducible expression vector PMDC7 was constructed,and Js MYB305,Js MYB86,Js MYB108 and Js LHY genes were transformed into jasmine stem callus,and the relative expression of Js BEAT1 and Js BEAT2 genes in jasmine callus was detected.The analysis showed that Js MYB305 was positive for Js BEAT1 in jasmine stem callus.The promoter has the strongest transcriptional activation effect,followed by Js MYB86;Js LHY has the strongest transcriptional activation effect on the Js BEAT2 promoter,followed by Js MYB108.In different plant systems,the jasmine Js MYB transcription factor has different activation levels of the Js BEAT1 and Js BEAT2 promoters,due to the regulation of other endogenous transcription factors in the plant.In summary,this article identified two key enzyme genes Js BEAT1 and Js BEAT2 for the synthesis of benzyl acetate in jasmine.The two enzymes have different substrate affinities,but both exist in the petals at the same time,controlling the formation of the floral component benzyl acetate..In addition,their upstream regulatory factors were discovered and showed inconsistent levels of regulation.This provides the possibility to further clarify the genetic regulation mechanism of jasmine floral fragrance,and also for the molecular breeding of jasmine with high "sweet and fruity fragrance" and the selection of new varieties with high fragrance.It laid the foundation for breeding,and at the same time provided alternative jasmine genes for the production of spice substances by bioengineering.