Molecular Regulatory Mechanism Of Secondary Laticifer Differentiation Induced By Coronatine In Hevea Brasiliensis

Natural rubber as well as oil,coal and iron are known as the four major industrial raw materials.It is also a kind of important strategic material and closely related to the national economy and people’s livelihood.More than 99%of natural rubber was produced from Hevea brasiliensis Muell.Arg.Secondary laticifers in trunk bark of rubber tree are directly related to the natural rubber production and differentiated from vascular cambia.Since natural rubber biosynthesis and storage occurrs in the secondary laticife,the number of secondary laticifers is the most important stracturnatural factor that ditermines rubber yield.Available data show that jasmonic acid and linolrnic acid,the precusor substance of jasmonic acid,are effective in inducing the secondary laticifer differentiation,which is indicative that the secondary laticifer differentiation may be mainly regulated by jasmonic acid signaling pathway in rubber tree.CoI1-JAZ-MYC module has proven to be pivotal in jasmonic acid signal pathway in Arabidopsis thaliana,and several gene family members related to the COI1-JAZ-MYC module have been cloned in the rubber trees.However,the molecular mechanism for the role of jasmonic acid signaling in regulatoffing the secondary laticifer differentiation remains largely unknown due to the lack of suitable experimental system.Coronatine(COR)is similar to the active forms of endogenous jasmonic acid.In the present study,an experimental system of COR-induced the secondary laticifer differentiation was built up and the molecular mechanisms for the regulation of jasmonic acid signaling on the secondary laticifer differentiation were analyzed by the methods of RACE,real-time PCR,suppression subtractive hybridization and comparative transcriptomics.The main results as follows:1.It was found that COR can effectively induce the secondary laticifer differentiation in H.brasiliensis in a dose-dependence manner with a minimal effective concentration of 10 μM.2.An experiment system of COR-induced the secondary laticifer differentiation iwas build up.The system is suitable for investigating the secondary laticifer differentiation since both weather and season had little affect on the secondary laticifer differentiation and what is more,the laticifer differentiation was highly consistent among different rubber shoots.3.The full-length cDNA sequences of ten number of MYC transcriptional factor family,designnted as HblMYC6 to HblMYC15,were obtained by the method of RT-PCR and RACE from the laticifer of H.brasiliensis.Bioinformatic analysis showed that all the ten putative proteins possessed a typical bHLH domain and nuclear localization signal,suggesting that they belonged to the MYC transcriptional factor family.4.Tissue specific expression and the expression pattern of 15 MYCs upon tapping,jasmonic acid,ethylene were analyzed by real-time PCR.Transcripts of nine members of HblMYC1-4,HblMY6,HblMYC7,HblMYC11,HblMYC14 and HblMYC15 were most abundant in latex,while that of HblMYC10 and HblMYC13 were very low abundance in latex,HblMYC10 was higher in root and bark,HblMYC13 was higher in bark,flower and root.The transcripts of HblMYC5,HblMYC8,HblMYC9 and HblMYC12 were highest in other tissues,of which,that of HblMYC5 was higher in fowler and endosperm,HblMYC8 was higher in root and bark,HblMYC9 was higher in bark,root,fowler and leaf,HblMYC12 was higher in fowler and leaf.Mong the nine members that were abundant in latex,four members,HblMYCl,HblMYC3,HblMYC4 and HblMYC7,were up-regulated while HblMYC2 was down-regulated by both tapping and jasmontes.The reverse pattern was right in respond to ethylene.Althouth the transcripts of HblMYC10 and HblMYCl3 were very low abundant in latex,the two members were up-regulated by tapping,but down-regulated by jasmonic acid and ethylene.The transcripts of HblMYC5 and HblMYC12 were in low abundance in latex,but up-regulated by all of the tapping,jasmonic acid and ethylene.It was noted that HblMYC12 had an intensive response to ethylene.The transcripts of HblMYC8,HblMYC9 and HblMYC13 were in high abundance in bark and root.The three members were up-regulated by tapping,jasmonic acid and ethylene only at the time interval of 8 h.In fect,HblMYC8 and HblMYC9 were down-regulated upon tapping,jasmonic acid and ethylene.in early time and recover after tapping for 5 days wheras HblMYC13 was only shortly up-regulated after tapping for 12 hours.Besides,HblMYC8 was always up-regulated but HblMYC13 was always down-regulated and HblMYC9 was down-regulated in middle time by ethylene.HblMYC1,HblMYC2,HblMYC3,HblMYC4 and HblMYC7 were probably related to regulate rubber biosynthesis in rubber tree,considering that they were either up-regulated or down-regulated by both tapping and jasmonates.5.The expression pattern of the 23 key genes which were related to CoIl-JAZ-MYC module were analyzed in the cambium region by the method of Real-time PCR with the experimental system,.The results shown that HbCOIl,HbJAZl-7 and HblMYCl-15 were up-regulated by COR in the cambia.Based on several parameters such as gene expression abundance,the timing of the expression,duration time of up-regulated expression and maximum up-regulated multiples in cambium and primary latex,HbJAZ3,HbJAZ4 and HbJAZ5 and HblMYC3,HblMYC7,HblMYC10,HblMYC 12 and HblMYC13 were seemed to be closely related to the secondary laticifer differentiation induced by COR in H.brasiliensis.6.By screening the SSH libraries related to secondary laticifer differentiation induced by COR in H.brasiliensis,287 unigenes were obtained from the forward and reverse libraries,and their differential expression was verified by the method of reverse Northern blot.Of which,the expression pattern of 20 unigenes with differential expression were analyzed by real time PCR.Based on the expression pattern,CAMTA,CDPK1 and CABP,which was in Ca2+ signaling pathway,and PGA,and EIF may be related to the regulation of the secondary laticifer differentiation.7.Transcriptome Sequencing of the cambium samples treated with COR were performed.A total of 50 548 unigenes and predicted 47 397 CDS were obtained.Of which,8646 and 7134 unigenes were respectively up-regulated and down-regulated genes by comparision of the unigenes from COR and water treatment at early stage and 7711 and 12113 unigenes were respectively up-regulated and down-regulated genes at the late stage.These differentially expressed genes were primarily enriched in the metabolic and plant hormone signal transduction pathways.8.A universal histone deacetylase inhibitor,trichostatin A(TSA)were effe ctive in inducing the secondary laticifer differentiation in a dose-dependence m anner..The full-length cDNA sequences of seven number of histone deacetylase gene family,designated as HbHDAl to 7,and three numbers of histine acetyl transferase gene family,designated as HbGCN5,HbHAT1 and HbHAT2 were o btained.Comparative analysis of the gene expression of the 10 genes between TSA and COR treatments by Real-time PCR was performed.According to the consistency expression patterns upon the two treatments,histone deacetylase oth er than histine acetyltransferase were related to the laticifer differentiation,and HbHDA6 may be mostly the member associated with the regulation of the sec ondary laticifer differentiation via histone acetylation.Taken together,by using the experiment system of COR-induced the secon dary laticifer differentiation,expression analysis of the 23 COI1-JAZ-MYC mod ule-related genes within cambium region by the method of Real-time PCR,Sev eral members of JAZ and MYC families were preliminary identified to be relat ed to the secondary laticifer differentiation.In addition,a large number of diffe rentially expressed genes produced by SSH and RNA-seq was obtained.Of int erenting,a histone deacetylase inhibitor,TSA was found to be very effective i n inducing the secondary laticifer differentiation,and a members of histone dea cetylase gene was preliminarily identified to be related to the laticifer differen tiation.All these data should be helpful for elucidating the molecular mechanis m for the regulation of jasmonate signaling on the secondary laticifer differenti ation in rubber tree.