Preparation Of Protoplast Of Rubber Tree Mesophyll And Establishment Of Transient Expression System For Immunity Detection

Natural rubber is one of most important strategic materials and its productivity was seriously affected by various of diseases. Because of many problems on genetic transformation system in rubber tree, the research on its functional genomics and plant pathology had lagged obviously.In recent years, more and more attentions have been paid for plants PAMPs-triggered immunity (Pathogen-associated molecular patterns triggered immunity, PTI) due to its broad-spectrum resistance. Little is known in PTI of rubber tree and no research approach has been established. In this study, a stable protoplast transient expression system of rubber tree has been established to study the responses of several disease-related genes, such as HbFER, HbMPK3, HbMPK6, HbPRl, HbPR5, to different PAMPs includng flg22, chitin and cell wall extraction(CWE). A quick and sensitive method was established for detecting PTI reaction of rubber tree by dual luciferase reporter system. This method can provide an effective research tool to study the function of rubber tree disease-related genes, analyze the effect of pathogen effector immune responses and isolation the components of disease-resistant signaling pathways in rubber tree. The main results were as follows:1. In the preparation and culture of rubber tree protoplast, light green leaves and5h for enzymolysis time are favour of higher yield and quality. The viability of protoplast cells could reach more than97%. Otherwise, WI medium was better than W5medium in protoplast culture.2. The conversion rate and the expression of exogenous gene in protoplasts of rubber tree were analysed by green fluorescent protein (GFP) and Western blot. The results showed that conversion rate of rubber tree protoplasts was about50%and the expression of the target protein could be detected after4h and before22h and be stable in6-18h.3. Based on the partial FER sequence fragment obtained through blasting in rubber tree transcriptome database, the full-length cDNA of homologous gene of rubber tree was cloned by RT-PCR and RACE, named as HbFER.4. The expression change of HbFER, HbMPK3, HbMPK6, HbPR1, HbPR5in rubber tree protoplasts after treatment with flg22, chitin and CWE were detected by RT-PCR. The results showed that flg22can inhibit the expression of HbFER and induce the expression of HbPRl and HbPR5. Expression of HbFER was down-regulated, while HbPR1was up-regulated by chitin. HbPR1was the only one with response to CWE and in up-regulation way. HbMPK3and HbMPK6didn’t answer to any PAMP. These results implied that HbFER, HbPR1and HbPR5, but not HbMPK3and HbMPK6, were related with rubber tree PTI, but the signaling pathways involved were different.5. Based on the promoters of HbFER, HbPRl and HbPR5fused to LUC reporter gene, the activities of LUC were detected to investigate the effect of different PAMPs on expression of these genes by dual luciferase reporter system. The results showed that the activity of HbFER::LUC was significantly inhibited by flg22, while the activity of HbPR5::LUC could be induced significantly by flg22and chitin. The activity of HbPRl::LUC could be induced significantly by CWE. These results suggested that HbFER, HbPR1and HbPR5can be used as marker genes in the innate immunity of rubber tree induced by different PAMPs.