Screening Of Candidate Genes For Transport Of Phenylpropanoid In Rosa Rugosa

Rosa rugosa‘Zizhi’is an important ornamental and economical horticultural plant.Its excellent resistance,multi-season flowering and volatiles characteristics make it has unique position in garden landscaping.Rose is rich in a large amount of aromatic substances and various trace elements,which has excellent beauty and detoxification function,and rose essential oil is also known as "liquid gold".Phenylpropanoids are one of the main components of rose flower color and fragrance and are synthesized by the phenylalanine metabolic pathway.ABC transporters are transmembrane proteins that are ubiquitous in nature and are involved in transmembrane transport of phenylpropanoids.Studying the transport mechanism of rose phenylpropanoids has positive significance for the improvement of rose color and scent quality.In this study,roses were used as the main research materials,and transcriptome analysis and gene co-expression network construction were used to screen out ABCG candidate genes related to the transport of phenylpropanoids.The major results are as follows:1.In this paper,we first screened 794 genes in the module with high correlation with Flower stage 4(S4)by transcriptome analysis and gene co-expression network in different periods of Rosa rugosa ‘Zizhi’petals.KEGG enrichment analysis of these 794 genes revealed that 82 genes were enriched in the phenylalanine pathway and involved in the synthesis of phenylpropanoids in rose.Secondly,using the hidden Markov model-based HMMER and sequence similarity-based Blast,the ABC transporter in Rosa rugosa ‘Zizhi’ was screened,and then the phylogenetic tree was construction of the ABC transporter with the reported Ph ABCG1 protein and related species.three candidate genes was screened: Rr ZZ＿11690,Rr ZZ＿40646 and Rr ZZ＿42661.Then,using Port Param,SOPMA and Swiss Model to analyze the protein structure of these three candidate genes,it was found that the protein structures of Rr ZZ＿11690 and Rr ZZ＿40646 were similar,and the protein structure of Rr ZZ＿42661 was quite different from the other two.Subsequently,using the TMHMM software to determine the transmembrane domain of the candidate ABCG transporter,presumably suggesting that these three proteins may be transmembrane transporter.the expression of these three candidate genes in the different flower development stages of rose was analyzed by real-time PCR.The results showed that the Rr ZZ＿11690 and Rr ZZ＿40646 genes had the highest expression during the S4 period,while Rr ZZ＿42661 was hardly expressed during this period.Finally,subcellular localization analysis of the transmembrane regions of these three candidate transporters revealed that they were all localized in the cell membrane,Therefore,we initially screened the ABCG candidate genes Rr ZZ＿11690 and Rr ZZ＿40646 related to the transport of phenylpropanoids.2.The PM transcription factor is a MYB transcription factor that was synthesized in the early stage of our research group to regulate the accumulation of phenylpropanoids.Through transcriptome analysis of PM transgenic rose(Rosa rugosa ‘Zizhi’),strawberry(Fragaria vesca)and tomato(Solanum lycopersicum),5418 differentially expressed genes were screened in rose,2789 differentially expressed genes were screened out from strawberry,and 822 differentially expressed genes were screened out in tomato.The GO enrichment of these differential genes found that transgenic strawberries and tomatoes differed greatly in the categories of molecular functions and biological processes,while transgenic roses differed greatly in three categories.KEGG enrichment analysis revealed that differentially expressed genes in all three transgenic species were enriched in the phenylpropanoid biosynthetic pathway.These results also indicate that PM transcription factors are indeed involved in the regulation of phenylpropanoid synthesis.Differential expression analysis of ABC transporters in PM transgenic materials revealed that 50 of the roses,17 of the strawberries and 2 of the tomato had significant changes.Rr ZZ＿11690and Rr ZZ＿40646 were down-regulated in transgenic roses.In Conclusion,we obtained the transcriptome data of different periods of rose petals,and correlated them by WGCNA to enrich the phenylalanine metabolic pathway during the rose flowering stage 4.The sequence analysis and preliminary experimental verified that Rr ZZ＿11690,Rr ZZ＿40646 and Rr ZZ＿42661 three candidate genes was selected..The mechanism of PM regulation and its regulation of ABC transporters proteins were inferred by transgenic PM plant transcriptome analysis.It provides new ideas and genetic resources for the study of rose flower fragrance.