Resistance Analysis Of The Code Region Of Pib Gene To Blast In Transgenic Rice Under Different Promoters

Blast and sheath blight and bacterial leaf blight were three main diseases of rice,rice blast was a fungal diseases and caused by ascomycete Magnaporthe grisea Barr [a sexual generations Pyricularia grisea Saco.] of rice (Herbert,1971), this disease has occurred almost every cultivation countries and regions of rice, if it had suitable conditions, it's easy to pop and cause disaster, and even erected,the way to solve this problem is to cultivate disease-resistant varieties, The gene engineering was an efficient methods which transport disease resistance gene into rice varieties, it has a very important theoretical significance and application value in breeding for disease resistance of rice.Pib gene is a blast resistance gene which has been cloned through map-based cloning (Wang ZX et al,1999), belonging to NBS-LRR resistance gene family. This gene was 10322bp, and contains three exons and four introns, the first two introns located in the region of translation initiation codon ATG upstream. It's encoding a peptide which had 1251 amino acid, in the NBS areas of N-terminal, containing Kinase la,2 and 3a and 17 repeat motif structure in LRRs areas of C-terminal, there exist two clusters which composed by eight cysteamine histidine residues, it was not found in other R genes, and the cloning of Pib gene which provided an material for the research of the relationship between resistance gene structure and function at the molecular level.According to the structural characteristics of Pib gene, we use the CaMV35S+Pib, CaMV35S promoter as well as Pib gene promoter to drive full-length code region of the Pib gene, vectors were each named pNAR701, pNAR704 and pNAR705. and to explore the blast resistance of Pib gene which under different promoters and explore the relations between the promoter and gene function of disease resistance as well as the values in breeding of rice blast resistance. At the same time, we use CaMV35S promoter to drive the positive/anti-sense fragments of the first and the third exon of Pib gene respectively, and each vector were named pNAR707/pNAR708 and pNAR702/pNAR703. To explore the relationship between the different fragment of Pib gene and blast resistance. To further clarify the function of Pib genes, we constructed a interference vector pNAR706 and driven by CaMV35S promoter, study the function of Pib gene under reverse genetics aspect.And use the Agrobacterium-mediated transformation Nipponbare of rice varieties, a total of 186 transgenic plants were obtained, pNAR701 had 61 plants, pNAR702 had 12 plants, pNAR703 had 45 plants, pNAR704 had 43 plants, pNAR705 had 25 plants, there were non obtained transgenic plants of pNAR706, pNAR707 and pNAR708. PCR and Southern blot analysis showed that: the gene has been integrated into the genome of of Nipponbare and all had single copy. The test of TO seed germination of hygromycin-resistant showed that most transgenic plants showed separation of 3:1, it indicating that hygromycin gene in the transgenic offspring is stable genetic.Northem blot analysis showed that:the Pib gene fragments which driven by different promoter are able to express in all transgenic plants, but it had differences in quantity.Real-Time fluorescence quantitative analysis showed that: the Pib gene could expressed in all Ti generation of transgenic plantlets of pNAR701-,pNAR703,pNAR704 and pNAR705, furthermore, the 701-1 plants have the highest quantity of mRNA, but the 705-2 was lowest in quantity of mRNA; it exist difference of the mRNA of target genes in different transgenic plants, such as 701-1,701-2 and 701-3, the reasons which caused the difference in same transgenic plants may be due to the differences in copy number of target gene.Through the resistance analysis to rice blast of T1 and T2 generation of transgenic plants in seedling, we found no significant difference of rice blast resistance under different types of promoters which drove the full-length code region of Pib gene, and all strengthen five grades than the control Nipponbare in resistance and show high resistance to ZB1 and ZG1 race. It had no significant differences between different transgenic lines in rice blast resistance level, apart from the ZB1 and ZG1 race had different pathogenicity in control Nipponbare, there was no significant difference in pathogenicity for all transgenic lines.