Study On The Regulation Of Flower Scent Metabolism By MiRNA167 And Its Target Gene ARF6 In Hedychium Coronarium

Hedychium coronarium is a perennial herb that belonging to zingiberaceae.Petal white,shape like butterflies which is also known as butterfly flower,and the fragrance of flowers is fresh,it is mostly used for garden ornamental and cut flower production.The main aroma compounds are terpenes and benzoic acid methyl ester which are induced by many factors.miRNA are small RNA regulatory molecules,usually 20-22 base lengths,which can inhibit one or more target genes at transcription or post transcriptional levels to regulate floral substances metabolism.In this study,we first screened the suspected miRNA with ARF6 as target gene by network database and Hedychium coronarium transcriptome comparison.Then the target gene ARF6 and miR167 combination was preliminarily determined according to the high throughput sequencing results.The ARF6 full-length and miR167 mature sequence were cloned by PCR.The expression of miR167 and ARF6 in flowers different developmental stage were analysised by qPCR,it preliminarily indicated the relationship between miR167 and ARF6 expression and their relationship with fragrance release of flower.the combination of miR167 and the target gene ARF6 was identified by tobacco CO transformation,and we studied the regulation of miR167 and ARF6 genes on the floral substances metabolism and terpene synthase genes by experiments that include short tandem target mimic,virus induced gene silencing,exogenous hormone tratement,subcellular localization and bimolecular fluorescence complementation.It provided a new way to explore the molecular mechanism of Hedychium coronarium flowers terpenoides metabolism.The main results are as follows:1.The lus-miR167 a and vvi-miR167 c mature sequence were most likely to be the upstream miR167 sequence of ARF6 according to ARF6 sequence alignment.miR167 mature sequence was found by high throughput sequencing and was cloned by Stem-loop RT-PCR,it was proved that Hedychium coronarium contains the same miR167 mature sequence as lus-miR167 a and vvi-miR167 c.full-length cDNA sequence of the ARF6 gene was cloned by RT-PCR.2.Sequence analysis showed that the cDNA length of HcARF6 is 2763 bp,encoding 920 amino acids,which contains B3(B3 DNA binding domain),ARF(Auxin response factor)and CTD(C-terminal domain)conserved domain.Phylogenetic analysis showed that HcARF6 with ARF17 from Musa acuminata subsp.malaccensis in Malaysia are a subfamily.All ARF6 of different species amino acid sequence alignment contain B3,ARF and CTD conserved domain by homology analysis,suggesting the ARF6 protein function is similar.3.Determination of flower scent release in the development process by GCMS,and the expression pattern of miR167 and ARF6 genes was analyzed by qPCR.The results showed that the volatilization amounts of the main floral materials eucalyptol,ocimene and linalool of Hedychium coronarium increased with flower opening and reached the highest peak at 8 h after flowering.The expression of miR167 was relatively high at the bud stage,maintained at a low level during the blooming period.While the expression level of ARF6 began to increase from 8 hours before flowering,which was basically consistent with the starting point of flower fragrance release.4.The tobacco CO transformation experiment showed that miR167 could bind to ARF6 and inhibit its expression,and HcARF6 was the target gene of HcmiR167.5.GUS staining experiments showed that the pOx transient overexpression system was suitable for the Hedychium coronarium flower.pOx-STTM transient overexpression interferes with miR167,compared with the control group,the expression of miR167 decreased by 69%,and the expression of ARF6 increased by 80%,floral material linalool synthase gene TPS1,TPS5 and TPS8 were increased by 201%,237% and 41%,the relative expression of ocimene synthase gene TPS3 increased by 78%,benzoic acid methyl ester synthase gene BSMT1 increased by 225%.The floral volatiles of linalool and methyl benzoate of the experimental group increased by 82% and 283% reaching a very significant level;alloocimene increased by 42% reaching a significant level;while eucalyptol and ocimene were also increased by 20% and 30%.The results further indicated that ARF6 was the target gene of miR167,and miR167 was a negative regulator of floral substances metabolism.6.ARF6 was silenced by the virus,compared with the control group,the expression of ARF6 decreased by 68%,floral material linalool synthase gene TPS1,TPS5 and TPS8 were decreased by 35%,65% and 39%,ocimene synthase gene TPS3 by 72%,benzoic acid methyl ester synthase gene BSMT1 decreased by 81%.The floral volatiles of linalool,ocimene and methyl benzoate decreased by 71%,51% and 11%,eucalyptol and alloocimene decreased by 53% and 73%.The results showed that,contrary to miR167,ARF6 was a positive regulator of floral substances metabolism.7.Hedychium coronarium flower was treated with exogenous hormone IAA,compared with the control group,ocimene and eucalyptol of the experimental group increased by 47% and 56% reaching a significant level,linalool,methyl benzoate and alloocimene were also increased by 39%,21% and 49%.qPCR results showed that the expression of miR167 decreased by 86%,and the expression of ARF6 increased by 63%.After treatment,the content of aroma compounds increased,and the changes of relative expression of miR167 and ARF6 after treatment were consistent with the changes after silencing miR167,the expression of miR167 increased,and the expression of ARF6 decreased.The results showed that miR167 and ARF6 were regulated by foreign IAA,and further proved that AFR6,as the target gene of miR167,antagonized each other to control the synthesis and release of floral fragrance.8.PEG4000 mediated subcellular localization of Arabidopsis protoplasts showed that ARF6 was located in the nucleus.9.Bimolecular fluorescence complementation experiments showed that ARF6 and PYL could be weakly interacted each other.Furthermore,according to previous studies in the laboratory,ABA receptor protein PYL interacted with MYC2-1 to affect the metabolism of floral terpenoids,suggesting that miR167-ARF6 might regulate terpenoid metabolism by PYL-MYC2-1 pathway.10.The pOx-MYC2-1 overexpression vector were transformed into tobacco leaves by agrobacterium mediated transformation with ARF6 and PYL bimolecular fluorescent complementary vectors.Experiments suggested that MYC2-1 might as a protein Auxiliary factor could enhance the interaction between ARF6 and PYL.Further more,it was speculated that MYC2-1 might be associated with ARF6 and PYL to form a three component complex to regulate the metabolism of terpenoids.