| Rose(Rosa rugosa Thunb.)is an important ornamental and economical plant in the world,its oil extracted from flowers is expensive and has high health care value.2-Phenylethanol is one of the main components of rose essential oil.It is of great theoretical and practical value to increase the content of 2-phenylethanol in rose and to cultivate new rose germplasm with high essential oil content through genetic engineering.Based on the preliminary research of our laboratory,The genes(RrAAAT and RrPPDCs)related to 2-Phenylethanol biosynthesis were cloned from the ’Tang Hong’ rose as the experimental material.Then,we transfered the genes which related to synthesis of 2-phenylethanol into Petunia hybrida.The transgenic plants were obtained,and the function of the related genes was verified.Our research will lay the foundation for culturing the new rose cultivars with high essential oil content.The main research results were as follows:1.The genes related to 2-phenylethanol biosynthesis were cloned.The results have shown that the full length of RrAADC is 1466bp with the start codon,the complete open reading frame(1266bp),termination codon,the 3’ non-coding region(200bp)and poly(A)tail(11bp),which encodes 422 amino acids,and the Genbank No.is MG820126.The full length of RrPPDCl is 2078bp with the start codon,the complete open reading frame(1818bp),termination codon,the 5’ non-conding region(71bp),the 3’ non-coding region(177bp)and poly(A)tail(12bp),which encodes 606 amino acids,and the Genbank No.is KY622034.The full length of RrPPDC2 is 2151bp with the start codon,the complete open reading frame(1773bp),termination codon,the 5’ non-conding region(43bp),the 3’ non-coding region(323bp)and poly(A)tail(12bp),which encodes 606 amino acids,and the Genbank No.is KY62203 5.2.Spatiotemporal expression of RrAAAT and RrPPDCs gene was studied.We used fluorescence quantitative technique to study the spatiotemporal expression of RrAAAT,RrPPDCl and RrPPDC2 in roses.The results have shown that the expression level of RrAAAT was increasing with the flower blooming and reached its peak in the withering stage.The expression level of RrPPDC1 also showed a rising trend,and the rising trend from budding stage to full open stage was relatively flat,and the expression amount increased rapidly in the withering stage,which was 4.47 times of the full open stage.From budding stage to full open stage,the expression level of RrPPDC2 gene showed a trend of rising and then decreasing and reached the peak in the withering stage.The expression level of RrAAAT was highest in pistil,following is calyx,next is stamen and receptacle.The lowest expression level was in petals.In stamen and pistil the expression level of RrPPDC1 was higher,next is petal but showed a similar expression level in calyx,receptacle and pedicle.The expression level of RrPPDC2 gene was the lowest in petals,and the highest expression was in pistil.3.The over-expression vectors of four genes were established and the functional verification was investigated.The over-expression vectors of RrAADC,RrPAR,RrAAAT and RrPPDC1 genes were constructed,and the over-expression vectors of four genes and the pCAMBIA1304 vector were transfected into Petunia hybrida by agrobacterium tumefaciens.After GUS staining and PCR detection,pCAMBIA1304,RrPAR,RrAADC,RrAAAT and RrPPDC1 transgenic plants were successfully obtained.It was observed that RrPPDC1 transgenic petunia flowers were larger.The aromatic substances were determined by GC/MS,we found that the volatilization of 2-phenylethanol in RrAADC transgenic plants and RrAAAT transgenic plants increased compared with the control.The content of 2-phenylethanol in individual transgenic plants was twice more than that of the control.There was no significant difference in the aroma volatilization of the RrPPDC1 transgenic plants compared to the control. |
