Cloning And Characterization Of JA Signaling Related Genes In Hevea Brasiliensis

Hevea brasiliensis Mull.Arg is the most important source of natural rubber.To harvest more latex,farmers generally tap the bark of rubber trees once every 2 days or 3,therefore,rubber trees become the most frequently wounded plants in the world.The latex biosynthesis and drainage are wounding responses of rubber tree,and Jasmonic acid(JA)is the main phytohormone that controls plant wounding responses,so JA may also regulate laticifer differentiation and the latex biosynthesis.JA signaling pathway in rubber tree,however,has not been identified,such as how JA regulates the laticifer differentiation and the latex biosynthesis are still unknown,thus the basic research on rubber tree hits a bottleneck,so do the technical improvements for latex yields.In order to reveal JA signaling in rubber tree,a dozen of JA-related genes were globally cloned and identified in this work,the main results are shown as follows:(1)By using bioinformatical methods,cDNA sequences containing conservative domains of JAZs were screened,thereafter,the full-length cDNAs of Hevea JAZs were cloned via in silico assembly.In this way,12 full-length JAZ cDNAs were obtained,of which 7 HbJAZ open-reading frames(ORFs)were successfully amplified through specifically designed primer pairs.According to the sequential analogues of these Hevea JAZ genes to Arabidoposis JAZs,they were termed as HbJAZ1,HbJAZ2,HbJAZ7,HbJAZ8,HbJAZ9,HbJAZ10 and HbJAZll,respectively.(2)The quantitative real time PCR(qRT-PCR)results showed that all the HbJAZs have higher expression levels in leaves than those in latex and bark,moreover,HbJAZ1,HbJAZ7 and HbJAZll showed the highest expression levels in leaves,whereas the HbJAZl expression levels in latex and bark is higher than other Yeast-two hybrid(Y2H)assays showed that all HbJAZs,except HbJAZ2,could interact with one another to form either homologous or heterologous dimers.(3)Through interactions with HbJAZl,three MYCs transcription factors,termed as HbMYC2,HbMYC3 and HbMYC4,and one Zincfinger protein were identified and cloned.These proteins showed very strong transcriptional autoactivities.GFP subcellular localization showed that HbMYC2 and HbMYC4 are localized in nucleus,suggesting that they are potential transcription factors.(4)All 23 different bHLH proteins were collected for sequence alignments and phy-logenetic tree analysis.All JAZs in interaction with JA-regulated bHLH transcription factors,including AtMYC2/AtMYC3/AtMYC4/TT8/GL3/EGL3/JAMYC2/JAMYC10/NtMYCa and three HbMYCs were clustered together.Furthermore,HbMYC2/HbMYC3 and HbMYC4 showed high sequence similarities with AtMYC2/AtMYC3 and AtMYC4,indicating that HbMYC2/HbMYC3and HbMYC4 are potentially involved in JA signaling pathway in rubber tree.(5)Results of Y2H showed that HbMYC2 could interact with HbJAZ7,HbJAZ8,HbJAZ10 and HbJAZ11,whereas HbMYC3 could interact with HbJAZl,HbJAZ7,HbJAZ8,HbJAZ9,HbJAZ10 and HbJAZ11,and whereas HbMYC4 interacted with HbJAZl,HbJAZ7,HbJAZ9 and HbJAZ11,suggesting that all HbMYCs could interact with HbJAZs via sequence specifities.(6)Results of the differential expression of genes showed that HbMYC2 and HbMYC3 were induced by wounding,whereas HbMYC4 was suppressed;exogenous JA upregulated the expressions of HbMYC2 and HbMYC3,but HbMYC4 was inhibited;The expression patterns of ABA treatment were similar to that of JA,which HbMYC2 and HbMYC3 were upregulated,while HbMYC4 was down-regulated.(7)Digital gene expression(DGE)analysis revealed that 3662 genes were up-regulated and 2039 genes were down-regulated one hour after JA treatment,of which the largest amount of diverse expression genes are involved in plant-pathogen interaction signaling pathway,however,the most significantly diverse genes are related with alpha-linolenic acid metabolism.Three hours after JA treatments,4257 genes were induced while 2511 genes were suppressed,most of which involved in metabolic pathways,especially in nitrogen metabolism.(8)Our previous works showed that HbMYCs could interact with G-box cis-elements.To further identify the target genes of HbMYCs,we generated a Hevea Cis-Elements Scan(HCES)software,by which G-box containing promoters were genome-widely scanned.The scanning results showed that there are 2800 Hevea genes containing G-box cis-elements in their promoters.(9)Y1H(Yeast one hybrid)assay revealed that HbMYC2,HbMYC3 and HbMYC4 could really bind the promoter of sugar transporter,ST2,and activate the expression of AbA to ensure the growth of transgene yeast stains on medium with AbA.The higher the AbA concentration in medium is,the stronger interactions between genes during Y1H tests are.Our data shows that the yeast strains co-transferred with one of HbMYCs and promoter of ST2 can grow on the medium with AbA of 800ng/mL.In summary,the results shown in this work laid a foundation for identification of the mechanism of JA-regulated wounding response and latex biosynthesis.It is of a great significance for the basic research on rubber tree to improve rubber yields.