| Natural rubber(cis-1,4-polyisoprene)is one of four major industrial raw materials and an extremely important strategic resource.Rubber tree is currently the main source of natural rubber.Treatment of the bark of rubber trees with ehephon(an ethylene releaser)has been a routine measure to increase latex yield,but the molecular mechanism behind the stimulation of rubber production by ethylene still remains a puzzle.Deciphering the enigma is of great importance for improvement of rubber tree for high yield.Natural rubber biosynthesis may follow the mevalonic acid(MVA)pathway and 3-hydroxy-3-metylglutaryl coenzyme A(HMG-CoA)reductase(HMGR)is thought to be the first rate-limiting enzyme in the MVA pathway.HMGRs belong to a small gene family,of which HMGR1 is mainly expressed in the latex of rubber tree and is involved in the biosynthesis of natural rubber.In this study,bioinformatics technique and expression analysis were used to identify the key regulatory elements of rubber tree HMGR1 gene promoter and the regulatory factors of HMGR1 gene expression.This will enhance the understanding of the regulation mechanism of natural rubber biosynthesis and help to stimulate the production of latex by using hormones or related active substances.1 De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 hours(E8)and 24 hours(E24)were performed.51,965,770,52,303,714 and 53,177,976 high-quality clean reads from E8,E24 and C(control)samples were assembled into 81,335,80,048 and 80,800 unigenes respectively,with a total of 84,425 unigenes and an average length of 1,101 bp generated.10,216 and 9,374 differentially expressed genes(DEGs)in E8 and E24 compared with C were respectively detected.The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate(IPP)biosynthesis pathway.In addition,up-regulated genes of great regulatory importance in carbon fixation(Calvin cycle)were identified.Therefore the rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber,instead of rubber biosynthesis perse,may be responsible for ethylene stimulation of latex yield in rubber tree.The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation.Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree.2 The HMGR1 gene promoter was isolated from the leaf DNA of Hevea brasiliensis RY 7-33-97 by polymerase chain reaction(PCR)method.The promoter sequence length of HMGR1 gene is 1460 bp.PlantCARE online prediction of the promoter cis element showed that in addition to some essential cis-elements such as the TATA box,CAAT box and so on,it also contains some hormone response elements such as two jasmonic acid(JA)-related elements,three ethylene(ET)-related elements,one abscisic acid(ABA)-related elements,salicylic acid(SA)-related elements and so on,and stress resistance-related cis-elements such as the thermal stress-related elements,anaerobic induction-related elements,light reaction-related elements and the endosperm induction-related elements,etc.These results suggested that the promoter of HMGR1 gene in Hevea brasiliensis is likely to be a tissue-specific and inducible promoter,and may play a regulatory role in the growth,development and stress response of Hevea brasiliensis.3 The plant expression vector P1301-H1-1460 was constructed by linking the rubber tree HMGR1 promoter sequence to the report gene beta glucosidase(GUS)and transferred into the model plant Arabidopsis thaliana.PCR detection showed that the plant expression vector P1301-H1-1460 with GUS gene was successfully transferred into Arabidopsis thaliana.RT-PCR detection showed that the plant expression vector P1301-H1-1460 with GUS gene could be transcribed in Arabidopsis thaliana.4 GUS expression levels in the P1301-H1-1460 transformed Arabidopsis thaliana plants grown for two weeks sprayed by methyl jasmonate(MeJA),ethephon and other were detected with real-time fluorescence quantitative PCR(qPCR)after 0 h,3 h,6 h,9 h,12 h and 24 h exogenous hormone treatments,respectively.The results showed that the expression of GUS after different time treatments with MeJA were significantly lower than those in untreated transgenic seedlings,and increased then reduced in a certain time range treatmented with MeJA.Therefore,rubber tree HMGR1 promoter might be an inducible promoter,exogenous hormones MeJA might down-regulated the expression of HMGR1 but ethephon might down-regulate the expression of HMGR1.5 A series of 5 'end sequence deletion expression vectors of such as rubber tree HMGR1 gene promoter P1301-H1-1290,P1301-H1-894,P1301-H1-593,P1301-H1-444 and P1301-H1-188 were constructed and transformed into Arabidopsis thaliana to analysis the effects of different promoter sequence fragments on the promoter activity. |
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