Effects Of Jasmonates And Its Signal Pathway Element HcJAZ And HcCOI1 In Fragrance Metabolism Of Hedychium Coronarium

Jasmonates are prevalently existed in the nature,widely distributed in tender plant tissues,flowers and vegetative organs of plants.Jasmonates are related to the plants growth development and their self-defence systems.The key factors of the jasmonates compound signaling pathway were discovered.Also,the regulatory mechanism of the COI1-JAZs-MYC2 complex was revealed.In addition,the regulation mechanism of the related factors in the pathway was well elucidated,showing that JAZ and COI1 play a vital role.H.coronarium is a perennial herb which has been classified as Zingiberaceae that can emit a fresh scent.In this study,H.Coronarium was used as the research object.RT-PCR,yeast-two-hybrid,Bi FC,subcellular localization,HPLC-MS/MS and GC-MS technology were used.The effects of endogenous jasmonates and exogenous jasmonates on the development of H.coronarium were interrogated,and the related key genes of Hc JAZ and Hc COI1 that regulate the fragrance of flower and jasmonates signaling pathway were studied.The purpose is to make a theoretical basis.The main results are as shown below :1.The Hc JAZ1,Hc JAZ2,Hc JAZ3,Hc COI1-1,Hc C OI1-2 and Hc COI1-3 were cloned using the RT-PCR methods.The full-length c DNA of Hc JAZ1,Hc JAZ2 and Hc JAZ3 contain putative ORF of 708 bp,780bp and 687 bp,encoding 235,259 and 228 amino acid residues respectively.The full-length c DNA of Hc C OI1-1,Hc COI1-2 and Hc COI1-3 contain putative ORF of 1764 bp,1728bp and 1761 bp encoding 587,575 and 586 amino acid residues respectively.Sequence alignment analysis showed that Hc JAZ contains conserved TIFY and CCT₂ domain,while Hc COI1 contains F-box,AMN1,LRR domain.Phylogenetic analysis showed that Hc JAZ1 and Hc JAZ2 had the closest ge netic relationship with At JAZ1 and At JAZ2,whereas Hc JAZ3 had the closest relationship with At JAZ9 in comparison with JAZ in Arabidopsis thaliana.Hc COI1-1 and Hc COI1-3 are homologous to Gh COI1 of Monocotyledon and Ac COI1 of pineapple,while Hc COI1-2 had partly homology with Mn TIR1.2.The yeast two-hybrid vectors of Hc JAZ and Hc COI1 full-length were constructed respectively.Yeast two-hybrid analysis showed that Hc JAZ2 and Hc JAZ3 could bind with transcription factors Hc MYB1 and Hc MYC2.Hc JAZ3 could bind w ith Hc COI1-2 protein and transcription factor Hc IAA2 through Jas domain.Hc COI1-2 protein could also be bound with Hc IAA2.3.The Bi FC fusion expression vectors were constructed with Hc JAZ3 and Hc COI1-2 full-length respectively,which were named Hc JAZ3-YCE and Hc C OI1-2-YN E.The results showed that Hc JAZ3 interacted with Hc MYB1,Hc MYC2 and Hc COI1-2 respectively.4.The GFP transient expression vectors were constructed with Hc JAZ3 and Hc COI1-2 full-length respectively.The construct was transformed to protoplasts of Arabidopsis thaliana that was stained with DAPI.The results showed that Hc JAZ3 was located in the nucleus and Hc COI1-2 was localized in plastids.5.The results of spatial and temporal expression analysis showed that the expression level of Hc JAZ3 in petals,roots and underground stems was relatively low,and the expression level in stems and leaves was very high.Hc COI1-2 was expressed in different tissues.The expression level in petals,roots and underground stems was relatively low,while the expression in stems and leaves was relatively high.Hc JAZ3 and Hc COI1-2 were higher in the green bud stage.The expression level of Hc JAZ3 reached a peak in the initial blooming period,while Hc COI1-2 reached the peak in the early bud opening period.Hc JAZ3 reached the trough in the period of full bloom,and Hc COI1-2 reached the troughin the early bloom period during the senescence period when there was a certain rising.Similarly,Hc JAZ3 showed a same pattern in H.coronarium whose expression is very low,but in H.coccineum Buch.-Ham,the expression is higher.Hc C OI1-2 in H.gardnerianum has the highest expression.6.Endogenous jasmonic acid of H.coronarium was extracted from different developmental stage.Then,the content of jasmonates in H.coronarium was studied by HPLC-MS/MS.The result illustrated that the content of endogenous jasmonates in H.coronarium has a significant upward trend.In the outer bract opening period,it started to rise and reach a small peak,and it further reached the peak in the full bloom period.Jasmonates content started to decline when going into the senescence period.7.Exogenous JA treatment starting from different time point to full bloom period in H.coronarium,different period of the floral change regulation is inconsistent,screening out the obviously changing which is in outer bract opening period and initial bloom period after treatment in H.coronarium,after treatment,1.8-eucalyptol growed about 19% and 56%.Benzoic acid methyl ester decreased by 33% and 10%.When treated in initial bloom periods,?E?-?-ocimene was increase 20%.In the outer bract opening period and initial bloom period of treatment,linalool creased by 23% and 8% respectively.That shows that exogenous JA treatment from different periods to full bloom period in H.coronarium have a certain impact on its floral release.The expression of Hc MYB1,a key transcription factor for the jasmonic acid pathway,increased by 2.2 and 3.2 times in both periods,while the expression of Hc MYC2 increased by 60% after treatment in outer bract opening period.In addition,the expression of Hc JAZ3 decreased by 35% and 63%,respectively.The expression level of Hc COI1-2 increased by 1 fold in outer bract opening period,but it did not change in initial bloom period.The relative expression levels of functional genes such as Hc BSMT2,which were involved in the regulation of the aroma synthesis of the propanal pathway,were almost unchanged after treatment in outer bract opening period,and it decreased by 15% in initial bloom period.The expression of Hc TPS gene,which is involved in terpene synthesis pathway,showed an increasing trend.The expression level of Hc TPS1 increased most among Hc TPSs.Hc TPS3 increased by 12% and 9% in both periods.The expression changing pattern of functional gene is correlated to flower synthesis,which were consistent with the results of GC-MS.