The Transformation Of Pepper Antifungal Protein, Thionin-like Protein And Other PR Genes Into Rice And Their Preliminary Analysis Of Blast Resistance

As the main of food crops in china, Rice play an important role of food security and sustainable agricultural development in the strategic resource. For a long time, rice blast, sheath blight, bacterial leaf blight and other fungal or bacterial disease severely always limit the yield and quality of rice. Cloning of resistance genes through genetic engineering combined with conventional breeding techniques to cultivate new varieties of resistant plants,which is an effective way in modern crop genetic improvement.In this study, combining with a strong inducible promoter LURP1((Late Upregulated in Response to Hyaloperonospora parasitica),the broad-spectrum resistance genes (Antifungal protein,thionin-like protein, cyclophilin-like protein, ferredoxin, non-specific lipid transfer protein) which were isolated from peppers,were transformted into Nipponbare rice. The transgenic plants were used to blast inoculation for further preliminary analysis of resistance to rice blast.At last, strong disease resistance of transgenic lines were selected, which lay a foundation of rice high yield .The main results were as follows:1.Using Gateway technology to build the plant expression vectors of antifungal protein, cyclophilin-like protein, ferredoxin, non-specific lipid transfer protein plant expression vector and sequencing,The results showed that the five PR genes of pepper were constructed into pMDC-32 plant expression vector successfully,and the target gene sequence completely consistented with the original sequence;2.We used the mature seeds of Nipponbare rice as materials, according to the Agrobacterium-mediated genetic transformation method, those five PR genes of pepper transformed into Nipponbare rice, and the preliminary conversion rate and the positive rate were counted. The results showed that the mature seeds of Nipponbare callus induction rate was about 80% -90% and the conversion rate of was about 9% -15%, in which,the antifungal proteins callus number at the beginning of number screening were about 263, the number of seedling differentiation culture were 31, the conversion rate was about 11.79%; the Thionin-like proteins callus number at the beginning of number screening were about 235, the number of seedling differentiation culture were 25, the conversion rate was about 10.64%; the Cyclophilin-like proteins callus number at the beginning of number screening were about 187, the number of seedling differentiation culture were 17, the conversion rate was about 9.09%; the ferredoxin callus number at the beginning of number screening were about 206, the number of seedling differentiation culture were 23, the conversion rate was about 11.17%; the Non-specific lipid transfer protein callus number at the beginning of number screening were about 157, the number of seedling differentiation culture were 17,the conversion rate was about 10.83%. The extraction of DNA for PCR validation were used to calculated the positive rate of transgenic rice,which we obtained 30 strains of antibacterial proteins positive plants,and the positive rate was about 96.77%,the positive plants of Thionin-like proteins were 25,and the positive rate was about 100%; the positive plants of Cyclophilin-like proteins were 17,and the positive rate was about 100%;the positive plants of Ferredoxin were 23,and the positive rate was about 100%;the positive plants of Non-specific lipid transfer protein were 15,and the positive rate wasabout 88.24%;Because of the Cultivation management,the pathogen infection and other factors, some transgenic seedlings were dead.Finally,the 28 Antifungal protein positive plants,The 23 Thionin-like protein plants, the 15 Cyclophilin-like protein positive plants,the 20 Ferredoxin protein positive plants,the 15 non-specific lipid transfer protein positive plants were transplanted into field for harvesting T1 generation seed;3.The T1 transgenic plants were inoculated with rice blast fungus guy1,and we analysised the gene expression of the antimicrobial protein, thionin-like protein, non-specific lipid transfer protein gene in transgenic plants by RT-PCR.At last we identied the resistance phenotypes of those transgenic plants. The results of RT-PCR analysis revealed that exogenous antifungal proteins, thionin-like proteins, non-specific lipid transfer proteins were differently expressed either under the condition of rice blast fungus inoculation or vaccination, but the expression were increased after inoculation. Compared to the wild-type Nipponbare plants, exogenous antifungal protein, thionin-like protein, non-specific lipid transfer protein gene in the transgenic plants had more resistant to rice blast in different varying degrees. These results indicated that exogenous pepper defense response gene increased the resistance of the rice blast fungus. In addition, the results further validated the results of previous studies: different plants containde a similar set of defense response gene.4.The results of arabidopsis LURP₁ promoter in the expression of rice, we found that the LURP₁ which played an important regulatory role in the resistance of the dicotyledonous model plant Arabidopsis can also be induced the expression of the rice resistiance genes, indicating that the cis-element in the activation of plant defense responses in monocotyledonous and dicotyledon were conservative.