Identification And Functional Elucidation Of Oleoresin Biosynthesis Genes In Masson Pine

Conifer oleoresin is a highly precious raw material for industrial sectors and a precursor for biofuels,it has important strategic value in national economic construction.At present,the demand for oleoresin in domestic and foreign markets is sharply increasing,and elucidation of the mechanism for oleoresin biosynthesis is very important for the breeding of high-yield germplasm.Although there are many studies on the oleoresin biosynthesis mechanism of conifers at home and abroad,the overall understanding of related metabolic and synthetic pathways has been very limited.Masson pine(Pinus massoniana L.)is one of the most important native species for oleoresin production in China,most of oleoresin in China still obtained from this tree species.In the current study,SMRT full-length transcriptome and Illumina RNA sequencing were used to analyze the transcriptome of high-and low-oleoresin yield germplasm,and screened the candidate genes significantly involving in high-oleoresin yield through the correlation between oleoresin yield and gene expression,then the function of the candidate genes was illuminated,which laid a theoretical foundation for molecular assisted breeding for high-oleoresin germplasm.The main results are as follows:(1)Identification of terpenoids biosynthesis genes in masson pinePacbio SMRT combined with Illumina RNA sequencing technology was used to sequence the transcriptome of high-and low-oleoresin yield germplasm in different tissues and different stages of masson pine.A total of 76,754 full-length transcripts were obtained.Through comparative analysis,the transcriptome library can be used as reference data for gene annotation,gene function analysis as well as genome research of other gymnosperms.A total of 105 transcripts implicated in the biosynthesis of terpenoids C5 precursor were obtained,including 30 transcripts encoding seven enzymes in the MEP pathway,50 genes encoding enzymes of the MVA pathway,as well as 10 transcripts encoding IDI genes,19 PT genes,19 TPS DEGs and 125 CYP450 DEGs.Also,many transcription factor families related to biosynthesis of terpenoids,e.g.,WRKY,MYC,MYB,b HLH,NAC and AP2/ERF etc were investigated.(2)Molecular assistant candidate indexes for high-oleoresin germplasm breeding in masson pineTranscriptome and metabolome analysis showed that there were significant differences in terpenoid skeleton biosynthesis and diterpenoids biosynthesis pathways between high-and low-oleoresin yield germplasm.Combined with q RT-PCR,the spatiotemporal specific expression of 16 genes with complete ORF sequences and related to terpenoids synthesis significantly up-regulated in trunk xylem and needle of high-oleoresin germplasm was analyzed.It was found that the MEP pathway may play a more important role in terpenoids synthesis in comparison to the MVA pathway,especially MCS and HDS genes.In addition to the DXS gene,the expression of other13 genes,namely four genes in the MVA pathway(AACT,HMGS,HMGR,PMK),four genes in the MEP pathway(DXR,MCS,HDS,IDI)and four genes in diterpene biosynthesis pathway(ent-CDS,CDS,LDS and ent-KO3)were stably and obviously up-regulated in high-yield clones than that in a low one.The Perason correlation analysis between oleoresin yield and the relative expression of these 16 key candidate genes showed that the expression of ent-CDS9,HMGR7,IDI7 and HMGS7 could be used as candidate indexes for the development of molecular assisted breeding highyield germplasm.Additionally,the NAC transcription factor was also significantly upregulated in high-oleoresin germplasm.Further study on the structure and function of these genes will accelerate the breeding process for high-oleoresin yield germplasm.(3)Cloning and functional verification of key genes for oleoresin biosynthesis in masson pineThe full-length ORF sequence of ent-CDS9,NAC,MCS4 and IDI7 were 1887 bp,2119 bp,726 bp and 708 bp,which were named Pment-CDS9,Pm NAC,Pm MCS4 and Pm IDI7 respectively.The deduced proteins were potentially located in chloroplast,and they do not have a signal peptide and transmembrane region.Except that Pm MCS4 is a hydrophobic protein,the other three are hydrophilic proteins.Terpene synthase gene Pment-CDS9 may be related to the synthesis of sesquiterpenes and diterpenes.After they were overexpressed in tobacco,the plant height and leaf size of transgenic tobacco lines increased.Except for Pm MCS4,the total chlorophyll content of transgenic lines of the other three genes increased.The key limiting-rate enzyme genes of MEP and MVA pathway(Tb DXS and Tb HMGR genes)and isopentenyl diphosphate isomerase gene(Tb IDI gene)in transgenic tobacco were significantly up-regulated than those in wild type.Interestingly,the expression of the type II DXS gene was significantly upregulated in Pm NAC transgenic tobacco,and the expression of the type I DXS gene was remarkably up-regulated in the transgenic lines of the other three genes.The content of terpenoids in leaf secretion of transgenic lines somewhat increased.And overexpression of Pment-CDS9,Pm NAC,Pm MCS4 and Pm IDI7 genes can regulate the synthesis of tobacco terpenoids.