Cloning Of And Preliminary Function Study Of OsPEN1 Gene In Rice

In recent years, most of the plant resistance genes(R gene) had been cloned, molecular mechanism of the plant resistance was clarified gradually, it promoted plant resistance research. Compared with the plant resistance research which was cotrolled by R genes, though considerable progress had been made in understanding of plant non-host resistance, the molecular basis of plant non-host resistance was still poorly understood. Plant non-host resistance was the most common form of disease resistance exhibited by plant against the majority of potentially pathogenic microorganisms. It was not controlled by a single plant specific resistant gene. It was not easy to lose the resistance along with the pathogenic microorganism mutation, because it's broad spectrum and durable resistance, it had significant agricultural utility.AtPEN1(AtSYP121), AtPEN2 and AtPEN3 were previously identified in a mutant screen and were required for effective resistance in Arabidopsis to the powdery mildew fungi (Blumeria graminis f. sp. Hordei, Bgh) and Erysiphe pisi. Presently, these three genes were identified which control entry success of non-adapted fungal pathogens into epidermal leaf tissue. AtPEN1 encoded a soluble NSF(N-ethylmaleimide-sensitive factor) attachment protein receptor(SNARE) domain and plasma-membrane resident syntaxin. Because SNARE proteins played a key role in vesicle trafficking in eukaryotic cells, It indicated that there is a vesicle-associated resistance mechanism preventing powdery mildew ingress, the requirement for SNARE proteins implied a role for membrane fusion in non-host resistance. It was reported that AtPEN1 in Arabidopsis and HvROR2 in barley played an important role in plant non-host resistance. Furthermore, AtPEN1 also played an important role in plant non-host resistance to wheat powdery mildew fungi (Blumeria graminis f.sp. trtici, Bgt) in our study. Homology comparison revealed a high homology among OsPEN1, AtPEN1 and HvROR2. All of the three genes contained a highly conserved Qa-SNARE domain. It was reported that Qb-SNARE protein, encoded by OsNPSN11, participated in rice blast resistance. Therefore, the biological function of rice OsPEN1 was thought to participate in resistance to rice disease resistance.The sequence of OsPEN1 gene was obtained through homology comparison of AtPEN1 by BLAST programme in NCBI database, OsPEN1 gene was cloned from rice and then OsPEN1-overexpressing and RNAi vectors were constructed and were transformated into rice, and then transgenic positive rice plants were detected by PCR, these transgenic plants were analyzed by RT-PCR and northern blotting. Finally, inoculation resistance appraisal of transgenic rice to Magnaporthe grisea, Xanthomonas oryzae pv. Oryzae and Rhizoctonia solani. The main results were summarized as below:1. Cloning of OsPEN1 gene. Due to OsPEN1 gene had no intron, a pair of specific primers was designed according to OsPEN1 gene sequence, PCR amplification was performed on rice Nipponbare DNA template, OsPEN1 gene was successfully cloned by PCR, the target fragments were obtained by agrose gel electrophoresis. After beening purified, the products were cloned into pMD18-T vector, the successful construction of recombinant plasmids were identified by PCR, enzymes digestion and sequencing.2. Construction of plant expression vectors. Enzymes digestion of recombinant plasmids of pMD18-PEN1 and expression vectors plasmids, and then the OsPEN1 fragments were cloned into expression vectors. At last, the recombinant plasmids pCAMBIA-OsPEN1 and pUC-OsPEN1 were identified by PCR reaction, enzymes digestion. And then the rice transformation mediated by Agrobacterium tumefaciens by using Oryza sativa L.ssp. japonica cultivars Nipponbare.3. PCR detection of transgenic plants in T₀ populations. 22 over-expression and 22 RNAi transgenic plants were generated respectively. Transgenic plants which are resistant to kanamycin were detected as positive by PCR analysis. In total, 20 transgenic OsPEN1 -overexpressing and 17 RNAi transgenic positive plants were obtained.4. RT-PCR analysis of OsPEN1 gene. RT-PCR analysis showed that the expression levels of OsPEN1 gene in over-expression transgenic plants were higher than these non-transgenic plants, the expression levels of OsPEN1 gene in RNAi transgenic plants were lower than these non-transgenic plants. It indicated that transgenic plants expressed normally, the expression levels of transgenic plants which were inoculated with Rhizoctonia solani were higher than these were not inoculated with Rhizoctonia solani, it demonstrated that the expression of OsPEN1 might be induced by Rhizoctonia solani.5. Northern blotting analysis of transgenic plants. Different expression levels of transgenic plants were obtained from 12 OsPEN1 -overexpression and 10 RNAi transgenic rice plants. Compared with non-transgenic rice plants, the expression levels of OsPEN1 -overexpression transgenic plants were higher than non-transgenic plants, and the expression levels of RNAi transgenic plants were lower than non-transgenic plants, it indicated the OsPEN1 gene had expressed normally in transgenic plants.6. Resistance identification of transgenic plants. Inoculated with Magnaporthe grisea, Xanthomonas oryzae pv. Oryzae and Rhizoctonia solani on transgenic and non-transgenic rice plants, compared with non-transgenic rice plants, most of the OsPEN1 -overexpression transgenic rice plants were resistant to them, most of the OsPEN1-RNAi transgenic plants were susceptible to them, the non-transgenic plants were intermediate, it exhibited the potential broad-spectrum disease resistance characteristics of plant non-host resistance. These results suggested the OsPEN1 gene might play an important role in disease resistance. It might participate in plant non-host resistance.7. Study on bioinformations. OsPEN1 contained a trans-membrane domain, 284-305 aa was a trans-membrane region, 212-274 aa was a Qa-SNARE domain. The Qa-SNARE domain of OsPEN1 and other family members were highly conserved in different organisms, they had high homology. Presumably, OsPEN1 might contribute to increase plant disease resistance through participation in resistance signal pathway.