| Red raspberry is a kind of medicinal and food homologous plant,which has the functions of delaying aging,inhibiting obesity,anti-inflammatory,antibacterial,anti-cancer and preventing heart disease.Terpenoids are an important class of secondary metabolites,which have important medicinal value and are effective medicinal components of many Chinese herbal medicines.In order to clarify the regulation mechanism of terpenoid metabolism during the fruit development of the ’Heritage’ red raspberry(Rubus idaeus L.),the red raspberry fruits of the four developmental stages was used as the material.Non-targeted metabolomics based on LC-MS was used to detect and analyze terpenoids in the development process,and transcriptomics based on RNA-seq sequencing was used to annotate the functional genes and transcription factors of terpenoids biosynthesis and metabolism in the development process.The expression levels of key enzyme genes and transcription factors in the terpenoid metabolic pathway were analyzed,and the interaction of related genes and transcription factors was analyzed by using yeast two-hybrid technology.Through comprehensive analysis of transcriptome and metabonomics results,the relationship between the types and contents of terpenoids and the key enzyme genes and transcription factors in the metabolic pathway during the development of ’Heritage’ red raspberry fruit was clarified.The main findings of this paper are as follows:(1)Using LC-MS-based metabonomics technology to analyze the types and content of terpenoids during the development of the red raspberry fruit of ’Heritage’.The results showed that 21 kinds of terpenoids were identified during the development of the red raspberry fruit.The contents of undecaprenyl diphosphate showed an upward trend,the contents of aucubin,obacunone,Buddledin A and typosterol showed an upward trend at first and then downward trend,and the contents of capsidiol,oleanolic acid,Barringtogenol C,Cucurbitacin P,Azekisaponin III,Propapyriogenin A2 and cholesterol showed a downward trend at first and then upward trend.The content of alpha-Terpineol acetate,Alectrol,Eupatoriopicrin,Niveusin C,Mascaroside,isocolumbin and Asparagoside A showed a decreasing trend,while Asiatic acid and β-Sitosterol showed a trend of increasing,then decreasing,then increasing.Except for Asiatic acid,oleanolic acid,β-sitosterol and cholesterol,other terpenoids were detected in raspberry fruit for the first time.(2)RNA-seq-based transcriptome sequencing technology was used to determine and analyze the genes and transcription factors during the development of ’Heritage’ red raspberry fruit.A total of 61843 unigenes were annotated,of which 13,906 unigenes were annotated in KEGG database.The comparison results of different genes between groups were as follows,Period 4 vs Period 3>Period 2 vs Period 1>Period 3 vs Period 2.These results indicated that the overall molecular changes during the development of red raspberry fruit from red to deep red raspberry were more obvious than those in other developmental processes.Annotate the differential genes obtained by group comparison in KEGG,and obtain 6 KEGG pathways related to the synthesis of terpenoids.They are terpenoid backbone biosynthesis,monoterpenoid biosynthesis,diterpenoid biosynthesis,sesquiterpenoid and triterpenoid biosynthesis,steroid biosynthesis and carotenoid biosynthesis.During fruit development,the expression level of ispG,a differential expression gene related to terpenoids,was firstly decreased and then increased,the expression levels of DXS.SQLE,ACAT.TPS14.GGPPS1 and MVD increased with fruit maturation and development.The five enzyme genes of IDI.NCED.NUDXI.HMGR and FDPS showed a trend of increasing firstly and then decreasing.A total of 905 unigeneswere annotated into 34 transcription factor families during the development of red raspberry,and the transcription factors related to terpenoid synthesis mainly included MYB,AP2/ERF,WRKY,NAC,bHLH and bZIP families.(3)The expression levels of key enzyme genes for terpenoids synthesis and transcription factors during the development of ’Heritage’ red raspberry fruits were analyzed.The results showed that there were 34 transcription factors associated with MVD,135 transcription factors associated with SQLE,48 transcription factors associated with IDI and 43 transcription factors associated with HMGR gene expression during fruit development.The transcription factors with highly significant positive correlation with the expression levels of the four genes were mainly distributed in the MYB transcription factor family containing the MYB domain,NAC transcription factor family containing the NAM domain,ERF transcription factor family containing the AP2 domain and WRKY transcription factor family containing the WRKY domain.(4)The expression levels of four key genes,MVD.SQLE,IDI and HMGR,which regulate the synthesis of terpenoids were studied by qRT-PCR.Among them,the expression levels of MVD and SQLE continued to increase from Period 1-green fruit to Period 4-deep red fruit,The expression levels of IDI and HMGR increased from Period 1-green fruit development to Period 2-yellow fruit development,and decreased from Period 2-yellow fruit development to Period 4-deep red fruit.The results of qRT-PCR were consistent with the trend of RNA sequencing results,which also proved the reliability of RNA sequencing results.(5)The total length of RuMVD and RuR2R3MYB genes were 927 bp and 1470 bp,respectively.Bioinformatics results showed that the two genes encoded 308 and 468 amino acids respectively,among which RuMVD was predicted to be an acidic and unstable hydrophilic protein,while RuR2R3MYB was predicted to be an alkaline and unstable hydrophilic protein.Both of them are nonsecretory proteins and may be localized in the cytoplasm.(6)The pGBKT7-RuMVD and pGADT7-RuR2R3MYB expression vectors were constructed and transformed into Y187 yeast.It can be grown on three-deficient medium and four-deficient medium,which indicated that there may be interaction between RUMVD and RUR2R3MYB.In conclusion,this study clarified the specific components and content changes of terpenoids in the fruit development process of ’Heritage’ red raspberry,The metabolic pathways of terpenoids during the development of ’Heritage’ red raspberry fruits were elucidated,and the genes and transcription factors that may regulate the synthesis of terpenoids during the development and maturation of red raspberry fruits were explored.The results provided a scientific basis for the further regulation and utilization of terpenoids in ’Heritage’ red raspberry fruit,and provided theoretical value for the development of functional quality and timely harvesting of ’Heritage’ red raspberry fruit. |
