Cultivate Transgenic Rice With Elictor-coding Gene PeaT1 And Antifeeding Gene XnAFP2

XnAFP2, an Antifeeding protein, is purified from Xenorhabdus nematophila var. pekinggensis stain CB6. PeaT1, a protein elicitor, which can promotes plant growth, improves drought resistance, and induces broad-spectrum disease resistance is derived from fungus, Alternaria tenuissima. To study XnAFP2 and PeaT1's functions in plants, N6, MS and NB mediums were adopted for tissue culture of Duoxi 1 scutellum, and N6 medium worked best in the Duoxi 1 scutellum culture. Based on this result, Antifeeding protein xnAFP2 from Xenorhabdus nematophila and elicitor-encoding gene peaT1 from Alternaria tenuissima were introduced into the genome of Duoxi 1 and Nipponbare by Agrobacterium-mediated method. And we confirmed the integration, transcription and expression by PCR, RT-PCR, Southern blot and Western blot in transgenic rice.Key results are as follows:1. The high efficient plant expression vectors pCAMBIA2300-35S-xnaFP2-OCS and pCAMBIA 2300-35S-peaT1-OCS were constructed wich contain enhancer and poly-terminator. The DNA constructs were verified by restriction enzyme digestion and sequencing, then the plant expression vector was transformed into Agrobacterium tumefaciens strain LBA4404 by freeze-thaw method.2. N6, MS and NB mediums were adopted for tissue culture of Duoxi 1 scutellum, and the effects of the three mediums on the tissue culture were compared and N6 medium worked best in the Duoxi 1 scutellum culture. Based on this result, Antifeeding protein gene xnAFP2 from Xenorhabdus nematophila was introduced into the genome of Duoxi 1, elicitor-encoding gene peaT1 from Alternaria tenuissima was introduced into the genome of Duoxi 1 and Nipponbare by Agrobacterium-mediated method. So far T2 progenies have been harvested.3. Transgenic plants were confirmed by polymerase chain reaction (PCR). The integration of xnAFP2 and peaT1 gene was further confirmed by Southern blot analysis, transcription was verified by reverse transcription polymerase chain reaction (RT-PCR) and protein expression in tobacco was detected by Western blot.4. The growth parameters in Duoxi1 and Nipponbare transforments were analyzed. It was found that the Characters according to Mendelian genetics separation principle.5. Transgenic plants with peaT1 at T2 generation were challenged with drought. It was found that the transgenic plants have strong drought resistance capability compare to non-transgenic plants. Also, the feeding of xnAFP2 transgenic rice to Chilo suppressalis revealed that the transgenic rice showed the insect resistance compare to non-transgenic plants.