Functional Analysis On Candidate Gene Of A Novel Rice Blast Resistance Gene Pita~2

Rice，as an important grain crops, occupies a pivotal position in the global foodproduction and consumption. Blast, one of rice disease, is extremely serious in all ofour rice area. From the long-term perspectives of environmental protection andsustainable development of agriculture, the cultivation and rational utilization of newdisease-resistant varieties to control rice blast victims is the most cost-effective andsafest means to the environment. Therefore, the cloning research for the new blastresistance gene has a very important practical significance on breeding for diseaseresistance. In order to clone the new blast resistance gene Pita~2from Japonica PiNo.4quickly and accurately, the candidate gene function were preliminary analyzed on thebase of the early fine mapping of Pita~2, the major research results are as follows:1) The specific fragments of three candidate gene Pita~2-1, Pita~2-3, Pita~2-4were cloned into an excellent plant interference expression vector pMCG161,respectively. The sequencing analysis verified that three RNAi expression vectorswere successfully constructed.2) The three RNAi vectors by Agrobacterium-mediated genetic transformationof rice were transferred into donor varieties PiNo.4for functional verification. About10T1Ri,16T3Ri and22T4Ri positive T0transgenic plants were identified. TheqRT-PCR analysis showed that the Pita~2expression level in positive transgenic plantswere down-regulated, respectively. T1transgenic plants will be further for rice blasttest.3) About20,000seeds of Nipponbare near-isogenic to Pita~2were mutagenizedby EMS and4200lines of M1seeds were harvested. About800lines were inoculatedwith rice blast fungus53-33, and the36lines were verified to be susceptible. In thenext phase, the M2plants need further identification resistant phenotype andmolecular detection.4) The isogenic line NB-Pita~2, NB of resistance gene Pita~2were regarded asexperimental materials, and the relative expression levels of genes WRKY45, JAmyb,SalT, HSP90, SGT, RAR1, PBZ, PR1b were analyed in Pita~2-mediated resistance response by real-time PCR technology after rice blast fungus53-33inoculation. Theseresults are aid to study Pita~2involved in signaling pathways.