Screening And Preliminary Analysis Of The Candidate Genes For Rice Blast Resistance Gene Pi-ta~2

Rice is one of the world's most important food crops,providing food consumption to nearly 60%of the world's population.Rice blast caused by Magnaporthe oryzae is one of the most serious diseases in rice,seriously affecting rice yield.Studying rice blast resistance genes and breeding disease resistance cultivars is the most economical and effective method for controlling rice blast.Due to the variation and diversity of Magnaporthe oryzae,resistant varieties tend to lose resistance after several years of planting,so it is important to mine new resistance genes.The novel rice blast resistance gene Pi-ta² was derived from rice cultivar PiNo.4 and was initially mapped in the centromere region of rice chromosome 12.In the previous study,5 candidate genes were obtained by physical mapping.The functional complementation vectors and CRISPR knockdown vectors of candidate genes were constructed.The positive transgenic plants of 3 candidate genes were obtained.This study is mainly to verify the candidate genes of Pi-ta² and conduct a preliminary analysis of its functions.The main progresses are as follows:?1?Genetic transformation experiments were carried out on the complementary vectors of the other two candidate genes?Pita²-Seq5?Pita²-Seq8?,and the transgenic plants were confirmed by positive identification.Pita²-Seq5 had obtained 2 transgenic positive seedlings,Pita²-Seq8 has obtained 31 transgenic positive seedlings.Positive identification and disease resistance studies were carried out on T₁ and T₂ transgenic plants of Pita²-Seq4,Pita²-Seq5,Pita²-Seq6,Pita²-Seq8 and Pita²-Seq9.The disease resistance result showed that ONLY Pita²-Seq4 had strong resistance to inoculated rice blast race,and the candidate gene Pita²-Seq4 is the resistance gene Pi-ta².?2?Using PiNo.4?the donor of the resistance gene Pi-ta²?as the experimental material,Nipponare,LTH and CO39 as the control,the expression levels before and after inoculation of rice immune marker genes WRKY45,PR1b,NPR1 and AOS2 were analyzed by qPCR,using two pairs of compatible and incompatible rice blast strains?13014 and 13015,15098 and 15102?.The results showed that the expression of OsWRKY45,OsNPR1 and OsAOS2 were up-regulated and the expression of OsPR1b was down-regulated after inoculation with compatible strains;the expression of OsWRKY45 was not significantly changed,but the expression of OsPR1b,OsNPR1and OsAOS2 were significantly up-regulated after inoculation with compatible strains.The above results revealed Pi-ta² has the difference in the rice immune signaling pathway in the process of susceptible and resistance,which laid a foundation for further study of its molecular mechanism.?3?Genetic transformation of PiNo.4 was carried out on five candidate gene CRISPR knockout vectors,and 16 transgenic plants of KO-Seq4,KO-Seq6,KO-Seq5,KO-Seq8 and KO-Seq9 were obtained.After positive identification and target gene editing,the results showed that three positive seedlings of KO-Seq5 and one of KO-Seq9 were edited.Further differentiation is needed to obtain more transgenic plants,providing important materials for further studying the function and the molecular mechanisms of Pi-ta².