Research On The Competition And Interaction Of HcTIR5,HcJAZ3 And HcIAA2 In Fragrance Metabolism Regulation By Ubiquitination In Hedychium Coronarium

Hedychium coronarium is a perennial herb of Zingiberaceae,Hedychium.It has beautiful flower type and attractive fragrance,and it is an ideal material for the research flower fragrance metabolism.The main aroma components of H.coronarium are1,8-cineole,basilene and agaritol.The synthesis of these substances is related to terpene synthetase,which is regulated by the upstream jasmonate and auxin signaling pathway.In this paper,by the methods of GC-MS,LC-MS,q RT-PCR,Co-IP and Western blot,the signal interaction of jasmonic acid and auxin signaling pathway and its effect on the floral fragrance release of H.coronarium were investigated.The main results are as follows:1.By the method of GC-MS the effect of Me JA,acetylsalicylic acid,IAA,PCIB and their mixture treatment on the fragrance release were investigated.Me JA and IAA treatment significantly increased the release of 1,8-cineole,basilene and aloe alcohol,and acetylsalicylic acid and PCIB treatment significantly decreased their release.Me JA+PCIB and IAA+acetylsalicylic acid treatments respectively showed there was no significant change in the release of above three aroma components.2.By the method of GC-MS agaritol release from three species of H.coronarium at different flowering stages were determined.Meanwhile,by the method of LC-MS JA and IAA content were measured.The results showed that in the green bud period,agaritol release in white ginger flowers and gold ginger flowers began to increase,and reached the highest point in the blooming period,then decreased in the senescence period,while that of agaritol in red ginger flowers in the six periods was almost zero.In the first three periods,the content of endogenous JA in H.gardnerianum and H.coccineum was lower,and increased in the green bud period,reached the peak in the full bloom period,and decreased in the senescence period,which was similar to the release of aloe alcohol in every period.However,JA content in the flowers of H.coronarium reached the peak in the half opening period,while it was very low in the other five periods.IAA content of in the flowers of H.coronarium increased in the grey white period,decreased in the half open period,increased and reached the peak in the full bloom period,and decreased in the senescence period;the IAA content in the flowers increase in the F5 period and decreased in the senescence period;IAA content in the flowers of H.coccineum tended to be stable.3.The bioinformatics of HcTIR5 was analyzed by NCBI and Clustal W.The results showed that HcTIR5 and HcCOI1 have high homology,and HcTIR5 may have similar functions with HcCOI1.4.The p EQA-GFP vector of HcJAZ3 and HcIAA2 without terminator and the p EAQ-HIS vector of HcTIR5 without terminator were constructed respectively.The transformation of Agrobacterium and the infection of tobacco were carried out.The interaction of HcTIR5 with HcJAZ3 and HcIAA2 was proved by immunocoprecipitation and Western blot.5.The ubiquitination of HcTIR5 to HcJAZ3 and HcIAA2 in tobacco was preliminarily verified by Western blot.The results that the bright bands could be detected by blank control and Me JA treatment in the leaf protein of tobacco transferred into HcJAZ3-p EAQ-GFP plasmid alone showed that HcJAZ3 did not degrade under the condition of no HcTIR5.The bright bands could also be detected by blank control,proteasome inhibitor MG132 treatment,Me JA and MG132 treatment,but only dark bands could be detected in the tobacco leaves treated by Me JA alone in the leaves,HcJAZ3 protein may be degraded by HcTIR5.In the leaves of B.tabacum which were transferred into HcIAA2-p EAQ-GFP plasmid alone,bright bands were found in both treatments,which proved that HcIAA2 could not be degraded without HcTIR5.In the leaves of B.tabacum which were transferred into HcIAA2-p EAQ-GFP and HcTIR5-p EAQ-HIS plasmid together,blank control,MG132 treatment,IAA and MG132 mixture treatment show lighter bands,while in IAA treatment,only the darker bands could be detected,which proved that HcIAA2 protein might be degraded by HcTIR5 in IAA treated tobacco leaves.6.Pcabs-γ virus silencing vectors with 300 bp conserved regions of HcJAZ3,HcIAA2 and HcTIR5 were constructed respectively.The petals of H.coronarium were infected by vacuum infiltration method,and analyzed by GC-MS and fluorescence quantitative PCR.The results showed that after silencing the HcTIR5 the release of 1,8-cineole,basilene and agaritol decreased,among which agaritol release reached a significant difference,HcTIR5 expression decreased significantly,and the expression of related genes HcMYC2,HcTPS1,HcTPS3 and HcTPS5 decreased.After silencing the HcJAZ3 the expression of HcJAZ3 and the related genes HcMYC2,HcTPS1,HcTPS3 and HcTPS5 increased significantly.The release of 1,8-cineole and aloe alcohol increased,while rollerene did not change significantly,the expression of HcIAA2 decreased significantly,the expression of HcMYC2 and HcTPS1 increased almost two times of control,the expression of HcTPS3 did not change significantly,and the expression of HcTPS5 increased significantly.