Analyze The Effect Of MeJA On Autophagy In Laticifers Of Euphorbia Kansui Liou Based On The ITRAQ Proteomics Technique

Methyl jasmonate(MeJA)is a ubiquitous plant hormone,which help plant resist to various biotic and abiotic stresses and has certain effects on plant growth and development.Euphorbia kansui Liou is a traditional Chinese medicine,mainly used to treat tumors,cough,asthma and schistosomiasis.The medicinal ingredients of E.kansui are mainly synthesized and distributed in laticifers.Recent studies have found that MeJA can induce the differentiation of rubber tree’s secondary laticifers and increase the yield of secondary metabolites in latex.In addition,it has been reported that autophagy is involved in the development of laticifers in E.kansui,however,at present,the effect of MeJA on the development of laticifers in E.kansui has not been reported in detail;the occurrence mechanism of autophagy during the development of laticifers in E.kansui is not clear.In view of this,E.kansui was taken as the object and treated with MeJA at different times,and then based on proteomics technology,the effects of MeJA on the development of E.kansui laticifers from the perspectives of molecular transcription level,protein translation level,and subcellular level was investigated;and the yeast two-hybrid technique was also used to investigate the interacting protein of ATG18 a which is related to the development of laticifers in E.kansui.The results obtained above might provide basic data for further investigation on the molecular mechanism of autophagy in laticifers of E.kansui and its response to MeJA.Specific research results are as follows:(1)MeJA promoted the expression of autophagy lysosome pathway and ROS pathway related proteins of latex in E.kansuiThrough proteomics based on i TRAQ technology,the latex proteins of E.kansui treated with MeJA were analyzed,and proteins related to the development of E.kansui laticifers were mined to analyze and explore the effect of MeJA on the development of E.kansui laticifers.A total of 509 proteins were identified in proteomics,including 297 annotated proteins and 29 differentially expressed proteins.These differentially expressed proteins are mainly involved in glycometabolism pathway,anti-pathogen mechanism,lysosomal autophagy pathway,ROS pathway and so on.Four proteins of interest: α-l-fucosidase,β-galactosidase,cysteine protease and Cu/Zn superoxide dismutase were selected,and the primers were designed for the genes of the above proteins,and the transcriptional expression of these proteins was analyzed by RT-qPCR.The results showed that the expression patterns of these proteins at the transcriptional level were consistent with the patterns of protein accumulation after MeJA treatment,MeJA promoted the expression of autophagy lysosome pathway and ROS pathway related proteins of latex in E.kansui.(2)MeJA promoted autophagy in laticifeirs of E.kansuiThe expression patterns at the transcriptional level of some autophagy related proteins:ATG1c,ATG3,ATG7,ATG8s and ATG18 s were analyzed by RT-qPCR.The results showed that the autophagy related proteins above were up-regulated at the transcriptional level after MeJA treatment,and reached a peak at 36 h after MeJA treatment,and then decreased,of which ATG18 a is particularly significantly up-regulated.In addition,ATG18 a is the only one autophagy-related protein that was found in the latex proteomic data of E.kansui in response to MeJA,and its accumulation was different at different treating time.Thus,the transcriptome sequence of ATG18 a was analyzed,obtained anti-ATG18 a antibody through gene cloning and prokaryotic expression,and then analyzed the effect of MeJA on autophagy in combination with the universal autophagy monitoring protein ATG8.Western Blot results showed that the accumulation of ATG8 and ATG18 a increased after MeJA treatment.With monodansylcadaverin(MDC)as probe,and combining with anti-ATG18a-alexa Fluor488/anti-ATG8a-alexa Fluor 488,E.kansui stems in the different development stages were observed under the laser confocal microscope.It was found that ATG8,ATG18 a and MDC can be colocalized in autophagic bodies of E.kansui laticifers,and the changing trend of fluorescence intensity was along with the development of laticifers,which is weak to strong to weak.Moreover,among the laticfers at the same development stages,the fluorescence intensity of ATG8 and ATG18 a in E.kansui stem treated with MeJA became stronger,which was shown as: MeJA36>MeJA48>CK.At last,the transmission electron microscopy(TEM)results also showed that the number of autophagosomes were increased significantly in laticifers of E.kansui being treated with MeJA,and mainly manifested as mitochondrial autophagy.(3)There was interaction between ATG18 a and Ra BD2 c in laticfers of E.kansuiThe yeast two-hybrid and Bi FC techniques were used to search for proteins interacting with ATG18 a in E.kansui laticifers.In E.kansui c DNA library,15 positive clones were screened,from which six genes including: 26S proteasome regulatory subunit S10B,Ra BD2c,60S ribosomal protein L37a,cysteine protease,extend factor Tu,60S ribosomal protein 23 were selected to conduct yeast rotary validation experiments and a false positive,60S ribosomal protein 23 was eliminated.Afterwards,three of the remaining five proteins,the 26S proteasome regulatory subunit S10B,RaBD2c and cysteine protease,were selected for BiFC verification.The final results showed that there was an interaction between RaBD2c and ATG18a.In summary,as an exogenous hormone,MeJA promotes autophagy in E.kansui laticifers and may promote mitochondrial autophagy by increasing ROS levels.ATG18a,an autophagy related protein with high protein content in E.kansui laticifers,interacts with Ra BD2c,which may be involved in the response of autophagy to MeJA,but its mechanism needs further study.