Expression And Function Analysis Of Activator Protein Gene PemG1 From Magnaporthe Grisea In Rice And Tobacco

Protein elicitors are important signal molecules that trigger plants disease resistance. Defence responses will be induced once the elicitors were recognized by acceptors in plants. Research shows that PemG1 activator protein from Magnaporthe grisea is able to promote seeds germination and seedlings growth, and also induce rice resistance against rice blast fungus. To study PemG1's functions in plants, pemG1 gene was transferred into rice and tobacco. The transgene's integration, transcription and expression were identified. Disease resistance and agronomic traits of transgenic plants were also studied. Key results are as follows:(1) Plant expression vectors were constructed. The maize ubiquitin promoter/octopine synthase terminator system and kanamycin-resistant gene nptⅡ(neomycin phosphotransfersⅡ) were used for constitutive expression of the pemG1 gene. DNA constructs were verified by restriction enzyme digestion and sequencing and then introduced into Agrobacterium tumefaciens strain AGL-1 by freeze-thaw method.(2) Performances of N6 medium, MS medium and NB medium in tissue culture were evaluated. Results revealed that NB medium was the best for culture of Nipponbare scutellum tissue. Then, pemG1 gene was transferred into Nipponbare and tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. Primary rice and tobacco transformants were obtained. So far T2 progenies have been harvested.(3) Transgenic plants were confirmed to be electropositive by polymerase chain reaction (PCR). The pemG1 gene integration was further confirmed by Southern blot. The transgene transcription was verified by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot. PemG1 protein expression was detected by Western blot. Genetic analysis showed that the transgenes were segregated normally in the progenies.(4) Differences of agronomic traits between primary transgenic rice plants and non-transformed Nipponbare control were compared. The average height of transgenic plants was 60.0 cm and that of control plants was 43.6 cm. Transgenic plants harvested 63.8 full seeds on average; control plants harvested an average of 42.6.(5) Transgenic rice plants of T0 and T2 generation were tested for resistance against rice blast disease by detached leaves assay, spores spray inoculation and lacto phenol blue staining. Results indicated that the transgenic plants increased resistance to rice blast fungus by 36～56%.(6) Transgenic tobacco plants (Nicotiana tobacum cv. Samsun NN) of T2 generation were challenged with TMV (Tobacco Mosaic Virus). In comparison with TMV-infected wild-type SNN control, transgenic plants reduced hypersensitive response lesions by 35～47%. Northern blot results revealed that accumulation level of pemG1 steady-state transcripts corresponds to the inhibition rate of local lesions.