Study On The Expression Profile And Function Of Floral Scent-related MicroRNA And Their Targets In Hedychium Coronarium

H.coronarium is a perennial herb of the ginger family,and is an important cut flower and garden application flower in tropical and subtropical regions.The flower volatile substances are mainly monoterpenes,sesquiterpenes and phenylpropane.miRNA is a type of non-coding small moleculeRNA with a length of about 21-24 nucleotides.It is a type of negative regulatory factor for plant gene expression.It mainly regulates the expression of target genes at the level of transcription or post-transcription.In order to study the effect of miRNAs on the synthesis of volatile substances in H.coronarium,the petals of three different developmental stages were used as materials,integrated with smallRNAs,degradome and transcriptome sequencing and quantitative real-time PCR,tobacco co transformation,short tandem target mimic,virus induced gene silencing,exogenous hormone treatment and GC-MS technology.This study explored the effects of HcARF8-miR167n and HcTIR1-miR393a on the metabolism of volatile substances in H.coronarium,and provided new ideas for exploring the molecular mechanism on the formation of flower fragrance.The main results are as follows:1.Hi Seq2500 sequencing platform was used to perform smallRNA high-throughput sequencing on the petals of F1,F5,and F6 stage in H.coronarium.After filtering out low quality,adapters,too long and short sequences,there are 14445245(F1-1),13148718(F1-2),14524493(F5-1),18086005(F5-2),12504265(F6-1),11637758(F6-2)clean reads.Through sequencing and analysis,171 conserved miRNAs and 32 novel miRNAs were obtained in H.coronarium.Hi Seq2500 sequencing platform was used to degradome sequencing on the petal mixed samples from the three periods of F1,F5,and F6 stage in H.coronarium.After removing the low quality,adapters,too long and short sequences,about 2.6×10~7clean reads were obtained.Using Cleave Land software to analyze mRNA degradation sites,a total of 102 mRNA degradation sites were detected,corresponding to90 target genes.After analyzing the annotation information of target genes in the degradome library,it is speculated that 7 target genes(including auxin response factor,E3ubiquitin protein ligase,gibberellin oxidase,SCL transcription factor,MYB transcription factor)may be involved in regulating flower fragrance substance metabolism.In order to verify the reliability of sRNA sequencing,10 differentially expressed miRNAs were selected,and q RT-PCR was used to verify that their expression levels were different in the three periods.At the same time,the expression levels of the target genes of two miRNAs were selected for analysis.It was found that the expression changes of the target genes in the three periods were exactly opposite to the corresponding changes of the miRNAs,indicating that the expression of these target genes was negatively regulated by miRNAs.2.GC-MS was used to determine the release of floral aroma during the petal development in H.coronarium.The results showed that the volatile contents of eucalyptol,ocimene and linalool increased with flower opening and reached the highest peak in bloom stage.The gene expression of HcTIR1-miR393a and HcARF8-miR167n was analyzed by q RT-PCR.The gene expression levels of hco-miR393a and hco-miR167n were consistent with the release of floral aroma substances,while the gene expression levels of HcTIR1 and HcARF8 were relatively high during bud stage and decreased during bloom stage.Then there was a slight increase in senescence stage.The results of tobacco co transformation experiments showed that hco-miR393a and hco-miR167n can target and inhibit the expression of HcTIR1 and HcARF8,respectively.It was further verified that HcTIR1 and HcARF8 are the target genes of hco-miR393a and hco-miR167n.3.Short tandem target mimic interfered with the expression of hco-miR167n.Compared with the control,the gene expression of hco-miR167n,terpene synthase TPS8,TPS10,and methyl benzoate synthase BSMT1 decreased by 78%,53%,30%,and 60%,respectively.The gene expression of HcARF8 increased by 88%.Floral aroma substance such as basilene,agarol,pinene and methyl benzoate decreased by 37%,36%,68%and 79%.After interfering with hco-miR393a expression,compared with the control,the expression levels of hco-miR393a,HcTIR3,TPS5 and BSMT1 decreased by 52%,50%,59%,and 40%,while the expression levels of TPS1,TPS10,and BSMT1 increased by 59%,36%,16%.Floral aroma substances such as ocimene,linalool,pinene,and methyl benzoate decreased by 41%,28%,57%,and 42%.The results further showed that HcARF8 and HcTIR1 are the target genes of hco-miR167n and hco-miR393a,and hco-miR167n and hco-miR393a may be positive regulators of floral aroma substance metabolism in H.coronarium.4.After gene silencing of HcARF8,compared with the control,the gene expression of HcARF8 and methyl benzoate synthase BSMT2 decreased by 53%and 26%.The terpenoid synthetase genes TPS1,TPS3,TPS5,and TPS10 increased by 51%,95%,107%,and 80%,respectively.Floral aroma substance such as linalool and methyl benzoate increased by48%and 84%.Ocimene and eucalyptol decreased by 20%and 23%.After gene silencing of HcTIR1,the gene expression of HcTIR1 decreased by 43%,and TPS1,TPS3,TPS5,TPS8,TPS10,and BSMT1 increased by 99%,111%,123%,66%,205%,and 91%.The gene expression of BSMT2 decreased by 27%.Floral aroma substance such as linalool,allo-ocimene and methyl benzoate increased by 111%,29%and 18%.Eucalyptol decreased by 19%.Contrary to the effects of hco-miR167n and hco-miR393a,HcARF8 and HcTIR1are negative regulators of floral aroma substance metabolism.5.After treatment with the exogenous hormone IAA,compared with the control,ocimene,linalool,eucalyptol and allo-ocimene increased by 54%,21%,46%and 91%.Methyl benzoate decreased by 47%.The gene expression of hco-miR167n,HcARF8,hco-miR393a,and HcTIR1 increased by 149%,16%,284%and 19%.After treatment with the exogenous auxin inhibitor PCIB,ocimene,linalool,eucalyptol,allo-ocimene and methyl benzoate decreased by 57%,33%,24%,100%and 99%.The gene expression of hco-miR167n,HcARF8,hco-miR393a and HcTIR1 decreased by 30%,50%,38%and 61%.These results indicate that hco-miR167n and hco-miR393a are regulated by exogenous auxin,and further proved that HcARF8 and HcTIR1 as target genes of hco-miR167n and hco-miR393a indirectly regulate the synthesis and release of floral fragrance in H.coronarium.