| Natural rubber is indispensable industrial materials and strategic materials, which is important for national defense and economic development. Hevea brasiliensis is the important source of natural rubber. The disease of rubber tree is one of the most important factors that lead to serious yield losses in rubber production. In order to study the rubber disease-resistant mechanism and find some pivotal rubber tree disease resistance genes, Transcriptome analyses of rubber tree after inoculation with Colletotrichum gloeosporiodes was performed to find genes involved in disease resistance. Two of these genes, named as HbCDPK and HbFRO, were amplified and their function were explored. The results are as follows:1. Calcium dependent protein kinases (CDPKs) is the key enzyme of Ca2+signaling pathways and play a role in plant disease resistance reaction. Rubber tree HbCDPK gene encodes a535amino acids peptide, which molecular weight is60.1kDa and the isoelectric point is6.75. HbCDPK contained a special S_TKc protein kinase domain and EF-hand calcium combined domain which were similar to CDPK families members. The results of RT-PCR showed that Colletotrichum gloeosporiodes, but not Oidium heveae Steinm, can induce the expression of HbCDPK in rubber leaves after infected with these two pathogens. The salicylic acid (SA) and jasmonate (JA), but not ethylene (ET) also can induce the expression of HbCDPK, indicating that HbCDPK was involved in SA-and JA-mediated signaling pathways. Moreover, the expression of HbCDPK was induced by flg22(a peptide of bacterial flagellin) and chitin (fungal cell wall components), which are typical PAMPs (Pathogen-associated molecular patterns) that can trigger PTI (PAMP-triggered immunity) in plant. Overexpression of HbCDPK caused accumulation of reative oxygen species (ROS) in rubber tree protoplasts. ROS was well known as one of the early signal of plant defence respond. It is speculated that HbCDPK can promote the accumulation of intracellular reactive oxygen species to activate rubber defense response. Subcellular localization indicates that HbCDPK located in plasma membrane of rubber tree cells. HbCDPK transgenic Arabidopsis was generated and the T1seeds were obtained. The phenotype of HbCDPK transgenic Arabidopsis is identified.2. Ferrir chelate reductases (FROs) family is one of the key enzymes which response to iron stress for dicotyledons and non-graminaceae monocotyledon. participated in the interaction of plant and pathogenic microorganisms. The FRO homolog of rubber tree, named as HbFRO, contains a2217bp open reading frame (ORF), encoding739amino acids peptide with Ferric_reduct domain, FAD_binding domain and NAD_binding domain, and10transmembrance domains. The results of RT-PCR showed that the expression of HbFRO was decreased significantly in rubber tree after inoculation with C. gloeosporiodes and Oidium heveae Steinm, suggesting that HbFRO was involved in disease resistance of rubber tree. SA and ET, but not JA, can increase the expression of HbFRO, indicating that HbFRO was involved in SA-and ET-mediated signaling pathways. Additionally, the expression of HbFRO can be induced quickly by flg22and chitin, indicating its possible function in PTI response. Overexpression of HbFRO couldn't accumulate ROS in mesophyll cell of rubber tree. Result of subcellular localization showed that HbFRO located on plasma membrane. The data above suggested that HbFRO plays roles in disease resistance through regulating SA and ET signaling pathway and involved in PTI of rubber tree.In addition, TO seeds of HbFRO transgenic Arabidopsis were obtained. Identification and function assay of transgenic lines is ongoing. |
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