Studies On Genetic Transformation Of HbHMGR1Gene Driven By Laticifer-specific Promoter PHEV2.1in Hevea Brasiliensis

Rubber tree is one of the most important tropical crops, the natural rubber is an important strategic resource. In recent years, with the rapid development of the national economy and national defense, Chinese self-sufficiency rate of natural rubber has been declining and import dependence has been rising. Suitable cultivation area for rubber tree is limited, however, expanded acreage to increase rubber production potential is extremely small. So developing improved rubber tree clones and research on improving the yield of rubber is more pressing. Biosynthesis of natural rubber is an important factor affecting the latex yield of Hevea brasiliensis. Specifically improving rubber biosynthesis key enzyme gene expression is possible to have a positive effect on enhancing rubber yield. Friable and embryogenic callus from anthers of rubber tree elite clone Reyan8-79was used as recipient in the present studies. Single factor experiment design to screen optimum precultivation time, Agrobacterium concentration, infection time, co-cultivation time, co-cultivation temperature, co-cultivation pH and co-cultivation AS concentration. After co-cultivation, ticarcillin was used to inhibit Agrobacterium, and sulfuric acid kanamycin was used to select resistant friable and embryogenic callus. Resistant embryoids and regeneration plantlets were induced. GUS staining and molecular detection (PCR and IPCR) proved that one transgenic callus line containing CaMV35S::NPTII-PHEV2.1::HbHMGR1-35S::uidA was obtained. The main research results are the following:1. Laticifer specific promoter PHEV2.1sequence of1852bp in length which had98.6%identity with the sequence reported by Pujad-Renaud et al (2005)(GenBank: AY247789.1) was amplified by specific primers and PCR method.2. The recombinant plasmid specifically expressing HbHMGR1in laticifer was constructed. Double digestion and PCR identification were implemented. It proved that the plant expression vector pCAMBIA2301-PHEV2.1::HbHMGR1had been successfully constructed.3. The single factor experiment results from the Agrobacterium tumefaciens-mediated genetic transformation showed that the optimum conditions for friable and embryogenic callus from anthers of rubber tree elite clone Reyan8-79were:precultivation0d, Agrobacterium concentration OD600=0.6, infection time8min, co-cultivation time4d, co-cultivation temperature26?, co-cultivation pH5.7, co-cultivation AS concentration150?m.4. The kanamycin resistant friable and embryogenic callus lines were identified by GUS staining.5lines were positive. IPCR method was used to identify the resistant friable callus line11, which confirmed that the T-DNA of the plant expression vector was successfully integrated into the genome DNA of the resistant callus line11. One transgenic callus line with35S::NPTII-PHEV2.1::HbHMGRl-35S::uidA was obtained.5. A large number of resistant embryoids were acquired from the transgenic callus line11. Two complete transgenic plantlets were obtained.