| In order to further explore the key enzyme genes in the terpene synthesis pathway of Narcissus tazetta var.chinensis,discusse the genetic basis of flowers fragrance of N.tazetta and the molecular regulation model of terpenes metabolism pathway.This study organized the petals and corolla transcriptome data of the flowering stage in Narcissus tazetta’Yunxiang’,screened and excavated important enzyme gene of terpenoid biosynthetic pathway,cloned IDI and HDR which are the key genes in terpenoids synthesis way,using qRT-PCR analysis the expression levels in the flowering process of N.tazetta,at the same time using headspace sampling method combined with GC-MS technology in qualitative and quantitative analysis of terpenoids volatile substances in N.tazetta petals and corolla and the aroma components of their parents’ ’BaihuaⅠ’ and’Huanghua Ⅰ ’ narcissus,Combining volatile steroids with transcriptome data analysis to discuss IDI and HDR genes.This study provides some genetic resources and theoretical basis for the research on the synthesis of terpenoids from N.tazetta.A total of 54,517,142、54,431,118、52,132,752 and 53,415,454 clean reads were produced and assembled into 59,597、64,746、60,654 and53,562 Unigenes by transcriptome sequencing of the petal and corolla in the flower bud stage and the flowering stage of N.tazetta.The resultingunigenes obtained a total of 52 enzymes related to 17 synthesis of tepenes by blastn re-annotation.After secondary screening,11 candidate genes related to terpene synthesis were obtained: AACT,HMGS,HMGR,DXS,DXR,HDS,HDR,IDI,GPS,GGPS,FPS,and their transcription levels were verified by qRT-PCR.The content of terpenoids in the corolla and petals of N.tazetta was quantitatively analyzed by headspace sampling method combined with GC-MS technique.The results showed that the main volatile components of N.tazetta were(E)-B-ocimene and(Z)-β-ocimene.The types and contents of aroma components in the corolla at flowering stage are more than those in petals,and they show an obvious increasing trend compared with the bud stage,while there is little difference between the two periods in petals.Comparing the aroma components of ’Baihua 1’ and ’Huanghua1’ during the flowering period,all of them contained higher content of basilene and trans-ocimene,while other volatile components differed greatly in species and content.It is speculated that basilene and trans-ocimene are the main aroma substances.IDI and HDR genes were cloned from N.tazetta by RT-PCR and named NaIDI and NaHDR respectively.The ORF of the two genes was858 bp and 1383 bp,respectively,encoding 285 and 460 amino acids.The results of biosignal analysis indicated that the proteins encoded by both were acidic and stable hydrophobic proteins,and the predictions were alllocated in chloroplasts.The sequence alignment showed NaIDI and N.tazetta var.’Jingzhanyintai’,N.tazetta var.’Huanghua’,papaya,grape and olive oil.The amino acid sequence similarity reached 97.19%,96.14%,81.58%,79.49%,and 83.46%,respectively.The amino acid sequence similarity between NaHDR and asparagus,oil palm,small fruit wild plantain,pineapple and rubber tree reached 88.79% and 83.96%,respectively.82.93%,81.21%,84.72%.Phylogenetic analysis showed that NaIDI is closely related to narcissus,asparagus and oil palm,while NaHDR is closely related to rice and asparagus.The results of real-time fluorescence quantitative analysis of NaIDI and NaHDR transcription levels in petal and corolla of N.tazetta at different flowering stages showed that NaIDI was significantly different in different flowering stages,and its expression level was lower in the flower bud stage and alabastrum stage,but significantly increased in initial flowering stage and ful-bloom stage.NaHDR showed an upward trend throughout the flowering process,and NaIDI and NaHDR were both more expressed in the corolla than in the petals,suggesting that NaIDI and NaHDR play an important role in the synthesis and accumulation of terpenes during the terpenoid metabolism. |
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