Cloning And Functional Validation Of Key Genes For L-Phenylalanine Biosynthesis In Rosa Rugosa

Rose(Rosa rugosa Thunb.)is a rare natural fragrant plant in the world.The rose essential oil extracted from its flowers has the reputation of "liquid gold" and is very popular in the market.2-phenylethanol is one of the main components of rose essential oil,and L-phenylalanine is the substrate for the synthesis of 2-phenylethanol.It is of great theoretical and practical significance to increase the content of L-phenylalanine in rose by genetic engineering,so as to promote the synthesis of 2-phenylethanol and improve the yield and quality of rose essential oil.Based on the previous research in our laboratory,the RrDHQ/SDH,RrCM1,RrCM2,RrODO1 and RrMYB114 genes related to the synthesis of L-phenylalanine in rose were cloned,and their spatiotemporal expression characteristics were analyzed.and transferred the genes related to the synthesis of L-phenylalanine into Petunia,a model plant,to verify its function,in order to explore the key genes regulating the metabolism of L-phenylalanine in rose in order to provide theoretical basis for molecular breeding of "high oil" rose in the future.The main results are as follows:1、Using the Rosa rugosa ’Pingyin yihao’ as the test material the key enzyme genes RrDHQ/SDH,RrCM1 and RrCM2 in the L-phenylalanine synthesis pathway,and two transcriptions RrODO1 and RrMYB114 genes regulating the synthesis pathway of phenylpropanoid were cloned.Among them,the ORF of RrDHQ/SDH gene was 1518bp,encoding 531 amino acids;the ORF of the RrCM1 gene was 633bp,encoding 210 amino acids;the ORF of RrCM2 gene was 801 bp,encoding 266 amino acids;the ORF of RrODO1 gene was 837bp,encoding 278 amino acids;the ORF of RrMYB114 gene was 732bp,encoding 244 amino acids.2、Using ’Zizhi’ and its bud variety‘Baizizhi’ as experimental materials,which have obvious differences in the content of 2-phenylethanol in flowers,the temporal and spatial expression characteristics of RrDHQ/SDH,RrCM1,RrCM2,RrODO1 and RrMYB114 genes in different flower development stages and different parts of flower organs of Rosa rugosa were analyzed.The results showed that:(1)With the opening of flowers,in’Baizizhi’,RrDHQ/SDH gene was significantly up-regulated,and RrCM1 was significantly down-regulated in general;The transcription factors showed a pattern of induced expression in a specific period.And the expression of RrODO1 was only significantly up-regulated in the initial stage,which may be related to the accumulation of L-phenylalanine synthesis precursors,and RrMYB114 was up-regulated expression during half-open and full bloom periods,which maybe a regulator of 2-phenylethanol precursor competition;In ’Zizlhi’,the structural genes RrDHQ/SDH and RrCM1 were only significantly up-regulated during the decay period,which was consistent with the peak period of the phenethyl alcohol content.However,in decline period,the RrCM2 decreased significantly.The up-regulated expression of transcription factor RrODO1 in the half-open period may also be related to the accumulation of L-phenylalanine synthesis precursors,and in general RrMYB114 was down regulated.(2)In gene expression patterns of different parts of floral organs,RrCMl has strong tissue specificity,mainly expressed in petals;RrODO1 has strong tissue specificity and is only highly expressed in torus of ’Baizizhi’ and pistil of ’Zizhi’;RrMYB114 has a certain tissue specificity,abundance is low in calyx and stamen,and abundance of pedicel and pistil is relatively high;RrCM2 is not expressed in the stamens of the ’Baizizhi’ flower stalk,and is expressed in the rest of the parts.Its tissue specificity is significantly different from RrCM1;RrDHQ/SDH have certain expressions in all parts of flower and have higher abundance in the peduncle of‘Baizizhi’.3、Four overexpression vectors of RrDHQ/SDH,RrCM1,RrCM2,RrODO1 and RrMYB114 genes related to L-phenylalanine synthesis in Rosa rugosa were constructed,and the genes was transformed into Petunia by Agrobacterium mediated leaf disk transformation.The results of GUS staining showed that the injured tissues and plants of Petunia were obtained.Compared with the wild type Petunia,the phenotype of the transgenic petunia was observed.It was found that the morphology and flower size of the transgenic petunia were smaller than those of the control GC-MS analysis of the floral components of the transgenic petunia showed that the content of the main aromatic substances of the transgenic petunia was higher than that of the wild type,especially benzene/phenylpropanoid The content of 2-phenylethanol in transgenic plants was about 10 times higher than that in wild-type petunia plants.